Xenopus model to study the effects of ketamine on development. We found that exposure to ketamine from pre-gastrulation (stage 7) to early neural plate (stage 13.5) resulted in disruption of neural crest (NC) derivatives. Ketamine exposure did not affect mesoderm development as indicated by the normal expression of Chordin, Xbra, Wnt8, and Fgf8. However, ketamine treatment significantly inhibited Zic5 and Slug expression at early neural plate stage. Overexpression of Zic5 rescued ketamine-induced Slug inhibition, suggesting the blockage of NC induction was mediated by Zic5. Furthermore, we found Notch signaling was altered by ketamine. Ketamine inhibited the expression of Notch targeted genes including Hes5.2a, Hes5.2b, and ESR1 and ketamine-treated embryos exhibited Notch-deficient somite phenotypes. A 15 bp core binding element upstream of Zic5 was induced by Notch signaling and caused transcriptional activation. These results demonstrated that Zic5 works as a downstream target gene of Notch signaling in Xenopus NC induction. Our study provides a novel teratogenic mechanism whereby ketamine disrupts NC induction via targeting a Notch-Zic5 signaling pathway.Ketamine is a potent dissociative anesthetic and the most commonly used illicit drug. Many addicts are women at childbearing age. Although ketamine has been extensively studied as a clinical anesthetic, its effects on embryonic development are poorly understood. Here, we applied the  Neural crest (NC), the vertebrate-specific pluripotent cell population, derives from the border region between the epidermis and the neural plate. NC gives rise to numerous derivatives including cranial cartilages, neurons and glia of peripheral nervous system, melanocytes, and heart outflow tract . AbnormaThe development of NC undergoes induction, specification, migration, and differentiation, which is tightly regulated by a well-orchestrated gene regulatory network (GRN). After germ layer separation, a set of transcription factors including Pax3, Zic1, Zic5, Msx1, Hairy2, Ap2α, and c-Myc are expressed at the border region between the neural plate and the epidermis. Such expression is induced by morphogens like Wnt, BMP, FGF, Notch and retinoic acid in a gradient sensitive manner. These border determination transcription factors further trigger the expression of a group of genes including Slug, FoxD3, Snail, Sox9, and Twist in the emerging NC cells, thereby accomplishing NC induction . After iThe Notch signaling pathway plays vital roles in early embryonic development , 1999. FXenopus models demonstrate a similar BMP-regulating mechanism of Notch pathways in NC development  were listed in Table 1.The cranial cartilage staining was performed as previously described . For immT-Leukemia cell lines Jurkat were cultured with 2 mM ketamine for 10 h and collected for WB. The Antibodies used were Notch-1 antibody , GAPDH . For Notch ubiquitin assay, 5 μM MG132 (Sigma) was added with or without ketamine into the media, Jurkat cells were then lysed and precipitated with Notch-1 antibody (1:50) and then bound with protein A/G (Santa Cruz sc-2003). Anti-ubiquitin (Santa Cruz sc-8017) antibody were applied to detect ubiquitinated proteins.Xenopus tropicalis Zic5 gene were cloned and linked with XhoI and MluI restrictive site into pGL3-basic to generate the firefly reporter constructs. The primers for different lengths of X. tropicalis Zic5 upstream regulatory sequences are: reverse primer XhoI: 5′-ccctcgagtgtctgcctcccaactct; -4042 forward: 5′-cgacgcgttgagatggcgagtaggct; -1950 forward MluI: 5′-cgacgcgttccttattagtgtatata; -532 forward: 5′-cgacgcgttgcacaactataggtctatt; -286 forward: 5′-cgacgcgtctccaaactttctacaagtg; -200 forward: 5′-cgacgcgtcagccagccaatcagaaaag; -200m forward: 5′-cgacgcgtttctaattaaggattgaaaagcgggcctcc; -186 forward: 5′-cgacgcgtgaaaagcgggcctcctgcc. To mutant canonical CSL binding site, mutant kit (Takara R401) as well as template construct pXzic5 -4042/-29 was applied according to the manufacture with primers: for 3950 site forward 5′-atgtattgcttcgaccttggcattttgg, reverse 5′-gtttctacattttccaaagttgtgcaaag; for 65 site forward 5′-ggacagacctacggaaggaataagc, reverse 5′-ctccagcgattggcagaaagcg.Different fragments (100 bp–4 kb) upstream of To measure the reporter activity in embryos, 12.5 pg constructed firefly plasmid, 2.5 pg renilla construct and 500 pg NICD mRNA or 500 pg LacZ mRNA (for control) were co-injected into both side of the dorsal blastomeres at the 4-cell stage. The embryos were then harvested at the neural plate stage (stage 15), divided into three groups (>10 embryos each group), lysed and analyzed using Dual-Luciferase Reporter Assay System (Promega).SHSY-5Y cell line was transfected with pcDNA4-NICD or pcDNA empty vector. Nuclear protein was extracted. EMSA was performed as described previously with minor adjustment . BrieflyXenopus embryos to 2 mM ketamine-MBS culture media from pre-gastrulation (stage 7) to early neural plate (stage 13.5), and then cultured in MBS media until harvest time. Upon ketamine incubation, pigment cells in trunk diminished and eyes exhibited developmental abnormities . Ocular pericytes located in retina ganglionic cell layer (GCL) and inner nuclear layer (INL) are derived from the NC  and loss of β-tubulin III expression in cell layer boundary  and lens  expression, indicating abnormal differentiation of lens and retina. At the tailbud stage, ketamine caused enlargement of intestinal system combining loss of enteric nervous cells , a mega-colon like (Hirschsrpung disease) phenotype  and marked reduction or even complete loss of all the cranial cartilage elements including Meckel’s (Mk), ceratohyal (ch), basihyal (bh), and branchial (ba) cartilages as demonstrated by alcian blue staining . These data clearly indicate that ketamine impairs NC development, causing neurocristopathies.Previous data indicates that 2 mM ketamine applied in culture yields approximately 0.4% accumulation, i.e., 8 μM inside embryo , which im the NC . FurtherSlug. The criteria for judging the expression pattern are based on two aspects, expression area and intensity. Compared to control, decreased expression area or signal intensity was determined as ‘reduced expression level.’ At the early neural plate stage (stage 14), when the NC has been induced, control embryos express a normal Slug expression pattern (in 32/32 embryos) . However, ketamine at a 0.36 mM culture concentration showed reduced Slug expression (36/37). The inhibition of Slug expression increases in a dose dependent manner. At a 2 mM ketamine concentration, Slug expression was severely reduced (41/41) . Morphogens from the mesoderm are critical for inducing the NC (in situ hybridization assay with mesoderm markers on the ketamine-incubated and control embryos prior to NC induction (stage 11.5) . There was no significant difference between ketamine and control groups in the expression pattern of four mesoderm maker genes including Chordin , Xbra , Wnt8 , and Fgf8  . These results suggest that ketamine does not affect mesoderm at the beginning of NC induction. These data indicate that ketamine blocks NC induction independent of mesoderm genesis.Induction is the first event in NC genesis. To investigate the effect of ketamine on NC induction, we examined the expression pattern of the NC specification gene g the NC . Thus, wXenopus embryos, can develop into almost any cell type given the proper signaling stimulation in vitro. In Xenopus animal caps, co-injection of Wnt8 and BMP4 truncated receptor (tBR) mimics endogenous NC stimulation and results in the expression of NC genes . In the presence of ketamine, Wnt8-tBR induced animal caps markedly reduces the expression of Zic5 and Slug . Zic5 is expressed in the NC area and plays an indispensable role in the induction process . The recovery NC gene expression may be due to the feedback of complicated GRNs . Consistent with RT-PCR data from animal caps and whole embryos, in situ hybridization results showed that ketamine treatment did not affect most NC induction-related genes including Fgf8 , Wnt8 , Msx1 , Pax3 , and Zic1  . However, the expression of Zic5 and the induction marker gene Slug were markedly inhibited by ketamine, with 97%  and 100% (n = 43) of embryos having inhibited expression respectively . Finally, exogenous injection of Zic5 mRNA at two-cell stage significantly rescued the ketamine-induced Slug inhibition  . Our data suggest that ketamine disrupts NC induction by inhibiting Zic5 expression.Neural crest induction requires high Wnt and intermediate BMP signaling input . At the NC genes . To deteNC genes . BMP tarNC genes , Dlx3, DNC genes  and AP2αNC genes , 2003 ar process . Since eZic5 inhibition during NC induction, we screened the most common NC induction morphogens including Wnt, Fgf, and Notch. Previous work showed that prospective paraxial mesoderm or Fgf8 induces Zic5 expression . However, ketamine exposure markedly reduced the expression of Notch target genes Hes5.2a, Hes5.2b, and ESR1 . Somite labeling with MyoD and Six1 transcripts showed slightly reduced signal  and a shortened embryonic body axis  that mirrors phenotypes seen in Notch-deficient embryos  and promoted Notch protein degradation . To ask whether Zic5 functions as a downstream target gene of Notch signaling, we assayed Zic5’s response to high and low Notch environment during NC induction in Xenopus embryos. Blocking Notch pathway with microinjection of Delta-stu mRNA . Activation of Notch signaling by injection of NICD .To investigate the upstream signaling pathways mediating ketamine-induced pression . Our resene Msx1  were nor embryos . To analstu mRNA  at the 4 of NICD  induced Zic5, we first examined whether the Zic5 gene contains Notch response element (NRE) in its upstream regulatory region. A 4013 bp containing sequence, between -4042 and -29 bp upstream of the translation start codon (designated as +1), for the X. tropicalis Zic5 gene was cloned into pGL3-basic to generate a firefly luciferase gene reporter construct pXzic5-4042/-29. Sanger sequencing confirmed the insertion of -4042 to -29 as identical to 117367902–117371914 of chromosome 2 . This 4 kb sequence contains two putative canonical Notch signaling CSL binding elements (GTGGGAA) in -65 to -58  and in -3950 to -3944  upstream of Zic5. To check the promoter activity and the involvement of these two CSL cis-acting binding elements, pXzic5 (-65M)-4042/-29 carrying the -65 binding site mutation to CCTACGG, pXzic5 (-3950M)-4042/-29 carrying the -3950 binding site mutation to CCTACGG, and pXzic5 (DM) -4042/-29 carrying both binding site mutations to CCTACGG were generated for reporter assays . NICD mRNA or LacZ mRNA (mock stimuli), and pXZIC5-4042/-29 or relative 4 kb insertion containing CSL mutant firefly constructs were co-injected into both dorsal blastomeres  at 4-cell stage. A Renilla luciferase construct was also co-injected into embryos as the normalization control. Upon NICD stimulation, pXzic5-4042/-29 (1.59 ± 0.23 RLU), pXzic5 (DM)-4042/-29 (1.51 ± 0.06 RLU), pXzic5 (-3950M)-42/-29 (1.49 ± 0.07 RLU) and pXzic5 (-65M)-42/-29 (1.37 ± 0.13 RLU) reveal significant high luciferase expression level (P < 0.05) compared with negative control (LacZ) stimulated pXzic5-4042/-29 (0.05 ± 0.01 RLU) group in Xenopus Embryos. Transcriptional activity exhibited no statistical difference among the 4 kb wild-type and the putative CSL-binding site mutant constructs . Our data suggest that there is functional NRE within region -4042 to -29, however, the two putative canonical CSL binding sites did not account for Notch-activated Zic5 transcriptional activation.To investigate the molecular mechanism underlying Notch’s effect on Figure 5B showed that NICD significantly activated the luciferase report plasmids containing -4042 bp (2.49 ± 0.62 RLU), -1950 bp (2.36 ± 0.45 RLU), -532 bp (2.73 ± 0.063 RLU), -286 bp (2.30 ± 0.62 RLU), and -200 bp (2.48 ± 0.42 RLU) upstream of Zic5 ORF with similar activation. A further deletion of 15 bp lead to a sharply reduced luciferase activity in pXzic5-186/-29 (0.187 ± 0.099RLU) (P < 0.05) . Therefore, the 15 bp sequence (5′-ACAGCCAGCCAATCA) located between -200 and -186 bp upstream of Zic5 was identified to contain a functional NRE. To confirm the results, we generated a new plasmid pXzic5-200M containing mutations in the NRE . The Zic5 upstream 200 bp reporter plasmid containing the 15 bp NRE was strongly activated by NICD (0.073 ± 0.0069 RLU), and the mutations in the 15 bp sequence to disrupt the NRE (0.007 ± 0.0009 RLU) abolished the activation of Zic5 by NICD .To identify the NRE, a series of deletion plasmids, covering various regions of this 4 kb fragment were cloned. Figure 5E). The competitor probe containing the same non-fluorescently labeled 15 bp sequence reversed the fluorescent binding signaling to background level, and a mutant probe had little effect to compete with the binding . These results unequivocally demonstrate that Notch activates Zic5 expression via the newly identified 15 bp NRE.Electrophoretic mobility shift assay (EMSA) was performed to further examine whether Notch signaling activation promotes the binding of the transcriptional factors to the 15 bp NRE. Nuclear proteins with or without NICD-transfected were extracted from SHSY5Y cells. NICD-transfection significantly increased the amount of the protein-NRE DNA binding complex (Ketamine is a dissociative anesthetic and the most popular club drug abused by youths for recreational purposes . Lately Xenopus (Xenopus embryos. Xenopus embryos exposed to ketamine also exhibited shortened somite-segmentation, a typical Notch signaling deficiency phenotype. The Notch signaling pathway has long been known to participate in NC induction (Xenopus NC induction. We also identified a 15 bp cis-acting element in the 5′ regulatory region of Xenopus Zic5 gene that binds to NICD to mediate Notch’s transcriptional activation of Zic5 gene expression. Our study further showed that ketamine enhanced ubiquitination of Notch proteins, and reduced Notch-1 protein level. These data suggest that enhanced degradation of Notch protein by ketamine may partly account for the phenotypes we observed in Xenopus, consistent with the similar phenotypes in Zebrafish (In this study, we discovered that early embryonic ketamine exposure specifically blocks NC induction via targeting Zic5. This effect on the NC is not due to the disruption of the mesoderm. Previous studies suggested the vital roles of Zic5 in NC genesis in mouse and Xenopus . Zic5 alXenopus . Howevernduction . In the nduction . Howevernduction ,b. In thebrafish . These rXenopus NC induction. Our study provides a novel teratogenic mechanism for ketamine whereby it disrupts NC induction by targeting a Notch-Zic5 signaling pathway.In summary, we discovered that Zic5 works as a downstream target gene of Notch signaling in YS, JL, and WS conceived and designed the experiments. YS, JL, CC, YXi, YL, and YXu performed the experiments. YS, JL, YXi, YXu, TL, and WS analyzed and contributed reagents/materials/analysis tools. YS, JL, and WS wrote the paper. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Agrobacterium-mediated transformation is an important research tool for the genetic improvement of cassava. The induction of friable embryogenic callus (FEC) is considered as a key step in cassava transformation. In the present study, the media composition was optimized for enhancing the FEC induction, and the effect of the optimized medium on gene expression was evaluated. In relative comparison to MS medium, results demonstrated that using a medium with reducing nutrition , the increased amount of vitamin B1 (10 mg/L) and the use of picrolam led to reprogram non-FEC to FEC. Gene expression analyses revealed that FEC on modified media increased the expression of genes related to the regulation of polysaccharide biosynthesis and breakdown of cell wall components in comparison to FEC on normal CIM media, whereas the gene expression associated with energy flux was not dramatically altered. It is hypothesized that we reprogram non-FEC to FEC under low nitrogen, potassium and phosphate and high vitamin B1. These findings were more effective in inducing FEC formation than the previous protocol. It might contribute to development of an efficient transformation strategy in cassava. Manihot esculenta Crantz) is grown throughout the tropics for its production of starchy roots. It is widely used in Africa and Asia for human consumption, animal feed, and as an industrial material [Cassava  at co-culture [http://www.nextgencassava.org/). The NextGen Cassava project aims to develop the human and infrastructure capacity at partner breeding programs and advance the full potential of cassava towards contribution to food security and livelihoods by breeding method knowns as genomic selection.Although these researches using efficient transformation system have been developed mainly by the use of model cassava cultivar 60444 due to its good capacity of FEC formation and its good regeneration capacity of embryogenic tissue, 60444 has not been cultivated by local farmers in Africa due to low yield, low nutritional quality and high sensitivity to viral and bacterial disease. The transformation system using other cultivars has been reported in a few cultivars but the following problems and/or optimization of several factors still remain: 1) which tissues, culture medium and genotypes should be used; 2) difficulty of the tissue preparation; 3) low regeneration efficiency of FEC , 21, 22.-culture –28. On tAgrobacterium-mediated transformation of cassava. After the induction of somatic embryos, the cultivation of cassava tissue on Gresshoff & Doy (GD) medium has been reported as the most effective method for inducing the formation of FEC. The use of nitrogen reduced medium, however, also seems to induce the formation of FEC after cultures have been first established on GD media [In the present study, an attempt was made to improve the protocol used to induce the generation of FEC, a prerequisite for GD media , 45. A gGD media . It is hManihot esculenta Crantz.) cultivar were obtained from the in vitro germplasm collection of the International Center for Tropical Agriculture (CIAT), Cali, Colombia. The in vitro plantlets were maintained by sub-culturing at 4–8 weeks interval on Murashige and Skoog basal salt (MS) with 20 g/L sucrose, 2 μM CuSO4, 2 mg/L glycine, 100 mg/L myo-Inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine·HCI and 0.1 mg/L vitamin B1, and 3 g/L gelrite which were adjusted to pH 5.8 using 1M KOH before autoclaving for 20 min at 121°C [in vitro plantlets were planted on 100 mL of fresh media, the in vitro plantlets were incubated at 28°C under a 16h light/8h dark photoperiod with the 40 μmol m-2 s-1. GD medium with vitamin [Plantlets of the ‘60444’ cassava , and 20–30 mm sections of single-node-stems were prepared. The single-nodal-stems were placed horizontally on petri dishes containing Murashige and Skoog basal salts  with 2 μM CuSO4, 10 mg/L of 6-benzylaminopurine (BAP), 2% sucrose, 10 g/L Noble-agar, 2 mg/L glycine, 100 mg/L myo-Inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine·HCI and 0.1 mg/L vitamin B1  (The formation of somatic embryos (SE) was induced at 2 weeks from axillary buds (AB) in a section of nodal stem of um, CAM)  and incuum, CAM) . The SEsum, CAM) .4, 12 mg/L picloram, 2% sucrose, 10 g/L Noble-agar, 2 mg/L glycine, 100 mg/L myo-Inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine·HCI and 10 mg/L vitamin B1  was induced from SE. SE were transferred to a modified MS basal salts with 2 μM CuSOium, FIM). FIM wasium, FIM). CIM-Ρ NAgrobacterium tumefaciens [gusA:gfp genes driven by a CaMV35S promoter, and a hygromycin resistance gene (HPTII) for selection. The bacterial culture was performed as described by Bull et al. [Agrobacterium suspension was prepared. After Agrobacterium suspension was collected by centrifugation , precipitation was re-suspended by 50 mL of FIM liquid media including 200 μM acetosyringone. The FECs were transferred to tea filter (30 mesh) in transgenic cassava plants was observed using a BX60 fluorescence microscope .2 of the small cut leaves. After the leaf samples were disrupted by ShakeMaster and zirconia beads (Hirata Corporation), genomic DNA was extracted from leaf chips of the transgenic cassava plants according to the protocol of Wizard Magnetic 96 DNA Plant System (Promega) using BIOMEK (Beckman Coulter) for the confirmation of gfp and hpt genes in genomic DNA of transgenic cassava. The genomic DNA was stored at -80°C until further use.The first or second healthy leaves from the top were used for the DNA preparation. DNA sample was extracted from 3 mmGFP and HPT genes in the transgenic lines of cassava was confirmed by PCR analysis using Ex taq polymerase (Takara) and the gene-specific primers listed in The presence of Multiplied FECs were transferred to several media and kept during 4 weeks at 28°C in dark. The total RNA from FEC was purified by use of the Plant RNA reagent (Thermofisher) following RNeasy Plant Mini Kit (QIAGEN). A 0.5 mL of Plant RNA reagent was added to about 0.1 g of fresh weight of finely powdered FEC tissue that was crushed by a Multi-Beads Shocker  and its mixture was mixed gently. After incubating for 5 minutes at room temperature, the supernatant was collected by centrifuging at 12,000 × g for 2 minutes at room temperature. The supernatant was transferred to a clean RNase-free tube. A 0.1 mL of 5 M NaCl was added to supernatant and tube was mixed by tapping. A 0.3 mL of chloroform was added to the supernatant and mixed thoroughly by inverting. The supernatant was collected by centrifugation at 12,000 x g for 10 minutes at 4°C to separate the phases. The upper aqueous phase was transferred to a clean RNase-free tube. After an equal volume of isopropyl alcohol was added to the collected aqueous phase, the mixture was incubated at room temperature for 10 minutes. After centrifuging the mixture at 12,000 x g for 10 minutes at 4°C, the precipitation was washed by 1.0 mL of 75% ethanol. After the solubilization of the precipitates in 40 μL of RNase-free water, the solution was treated with DNase I (10 U)(Takara) and RNase inhibitor (40 U) (Nippongene) in 50 μL of reaction at 37°C for 20 min. The 50 μL of solution was added to the 350 uL of RLT buffer in RNeasy Plant Mini Kit (QIAGEN) and the RNA preparation was performed as described by manual procedure. The total RNA quality was evaluated by electrophoresis with the bioanalyzer system . The total RNA was stored at -80°C until further use.2 ratio for display and analysis. The normalized signal intensity of transcripts for each experiment was used for the statistical analysis. An analysis of variance test was conducted to determine the effect of treatment medium (CIM vs. CIM-Ρ NPK) on the level of FEC formation. Changes in gene expression were statistically analyzed by the unpaired t-test for two groups. A false discovery rate  was calculated based on an unpaired t-test [Manihot esculenta microarray; Samples, GSM2394462, GSM2394463, GSM2394464, GSM2394465, GSM2394466, GSM2394467, GSM2394468 and GSM2394469.The total RNA purified from FEC was used to evaluate gene expression with a cassava DNA oligo-microarray that included more than 30,000 probes as described by Utsumi et al. . The pred t-test . The infhttp://primer3.sourceforge.net/). The PCR cycling conditions were as follows: 95°C for 20 s for initial denaturation, followed by 45 cycles of 95°C for 5 sec and 60°C for 30 sec. The specificity of the PCR amplification was evaluated with a melting curve analysis (from 55°C to 95°C) of the band pattern of the amplification product after the final cycle of the PCR. Each plate also incorporated a no-template control. Probes specific for the transcription factor SPN1  were used as a reference. The qPCR data was analyzed with the ΔCT method using the reference gene. For each sample, the mRNA levels of target genes were normalized to that of the reference.First-strand cDNA synthesis was performed using ReverTra Ace qPCR Master Mix with gDNA Remover . After denaturing 1 μg of total RNA at 65°C for 5 min, 4 μL of DN Master Mix was added to the total RNA in a 16 μL total volume. The reaction was incubated for 5 min at 37°C to remove the genomic DNA. The reaction was added to 4 μL of RT master mix and incubated at 37°C for 15 min. The RT reaction was stopped by heating for 5 min at 98°C. The first-strand cDNA preparations were stored at −30°C until use. The qPCR analysis was performed with a StepOne Plus Real-Time qPCR System  using first-strand cDNA preparations containing cDNA, 5 μL of Fast SYBR Green Master Mix , and 0.1 μM of both forward and reverse primers in a 10 μL total volume. The gene-specific primers used for qPCR are listed in All data are represented as means±SD from at least three biological experiments. Statistical analysis of means was assessed by analysis of variance using StatPlus 5 pro . Data for the optimization experiments was analyzed using One-Way ANOVA and differences among means were analyzed by Scheffé’s method at a 95% confidence level (p < 0.05) .2 enhanced somatic embryogenesis as described by Li et al. [4 and NO3) and SH basal salts were unstable condition for formation of organized embryogenic structure (OES), whereas GD media was the best media with regard to FEC induction and ½ MS and SH also gave a suitable FEC induction [Although GD medium has been used in various studies as the standard medium for FEC induction , the indi et al. . Practici et al. , they renduction . The fre3 and Ρ NH4) provided the highest frequency of FEC formation. The loss of either ammonium type-nitrogen, nitrate type-nitrogen, boron or zinc from CIM negatively affected the frequency of FEC production. Chloride was included in range of 0.6–3.0 mM in all media used in this study. Subsequently, potassium and phosphate were removed from the MS (Ρ NO3 and Ρ NH4) medium, and the frequency of FEC formation was evaluated though the difference was not statistically significant  on gene expression in FEC. Total RNA isolated from FEC cultured on CIM or CIM-Ρ NPK was hybridized to the microarray and differentially expressed genes (DEGs) were identified. All microarray data were applied on the principal component analysis (PCA) and the distribution of gene expression by histogram . PCA shoThe gene ontology (GO) analysis  and a PaMapMan metabolic pathways altered in FEC cultured on CIM-Ρ NPK media, relative to CIM media, are presented in 1  were induced in FEC cultured on CIM-Ρ NPK, relative to their expression in FEC cultured on CIM. PPCKs play a key role in the control of plant metabolism by the phosphorylation of phosphoenolpyruvate carboxylase . PEPC is mainly involved in the conversion of phosphopyruvate in glycolysis to oxalate in the TCA cycle. . Vitamin1 . Results1  by qRT-PCR . The expAgrobacterium  the acterium , 5) a seacterium , 6) an iacterium , 7) a shacterium  and 8) aAgrobacterium and FECs and the addition of hygromycin to CEM media in the process of root and shoot formation [Agrobacterium and FECs, the tea filter (30 mesh) was used for the collection of FECs, the conventional infection and the removal of excess Agrobacterium  or 14.8 (estimated as 5 independent experiments), respectively. We collected total 2,709 cotyledons (including non-transformants and transformants) from total 31.6 g fresh weight of FEC cultured on FIM. Among 2,709 cotyledons, 1,020 survived on CEM with hygromycin and finally 465 plantlets were regenerated. The percentage of regeneration efficiency was 45.6% . This was consistent with that of Bull et al. [In this study, we developed an improved medium for the efficient production of FEC in cassava, which is an essential need for the genetic transformation of cassava. Relative to the standard medium that is currently used, and other mediums that were evaluated, whereas the frequency of FEC formation was improved by culture on a nitrogen, phosphate, and potassium-reduced medium (CIM-Ρ NPK) and the non-FEC growth was inhibited on nitrogen, potassium and phosphate-reduced medium. Additionally, the frequency of FEC formation was also increased by the addition of excess vitamin B1 to the CIM-Ρ NPK medium (FIM). It is hypothesized that the reduced availability of nitrogen, potassium, and phosphate, and the use of excess vitamin B1 provided conditions favoring FEC induction by inhibition of non-FEC growth and promoted the structural changes of cell wall and recover of energy flux by nutritional changes that helped to maintain FEC structure.As reasons that frequency of FEC production on FIM was increased, we think: 1) the process of FEC identification was easier by inhibition of non-FEC growth, 2) structural changes of FEC in cell wall might be promoted by decrease of nutrition in media. In this article, we observed that the non-FEC growth on decreasing nitrogen, potassium and phosphate was inhibited. The non-FEC shows in the process of rapid cell division in comparison to FEC and SE and might consume more energy . On the 2 was found to promote SE formation but not FEC formation in cassava [Many studies have been conducted in cassava to increase the formation of SE and FEC in order to improve the efficiency of cassava transformation. High concentrations of CaCl cassava . An incr cassava , 16. Als cassava –28. The  cassava . In this cassava .Agrobacterium inoculation with FEC. Practically, transgenic lines could be obtained within 6 months after Agrobacterium inoculation in most cases. Taylor et al. (2012) obtained average 22.0 transgenic plants per cm3 of settled cell volume (SCV) of FEC of model cultivar 60444. When one gram of FEC was converted to 0.998 g per mL of FEC volume , nevertheless the method of regeneration process is similar to other method, indicating that the quality of FEC was not changed between FEC on media in this report and FEC on GD media.For the transformation efficiency, transformation efficiency by our protocol was not dramatically decreased compared with that by Taylor’s method . We obtaAgrobacterium-mediated transformation using 100 clusters of FEC for model cultivar 60444 [Agrobacterium inoculation with FEC [Agrobacterium infection and washing might be effective for improvement of cassava transformation because it can collect the smaller size of FECs. On the other hand, cost management must be also considered. All chemicals for preparation of FIM are obtainable at chemical companies independently. Therefore, we wish that cost for media preparation is reasonable but it might be dependent on country situation.Bull et al. (2009) prepared 50 transgenic plants by ar 60444 . Zainuddar 60444 . Chetty ar 60444 . The onear 60444 . Nyabogawith FEC . Althougwith FEC  and/or tAs current situation, it is one of important research to develop transformation methods for farmer- and industry-preferred landraces and cultivars for deployment and adoption of transgenic cassava in the field. Therefore, although we have attempted a transformation using Asian-elite cultivar KU50, we found that there is problem for FEC induction from the cultivar. It is that FEC formation was observed and the frequency of FEC induction was improved in comparison to GD media but the ability of FEC multiplication was quite low. To develop the cassava breeding in Asian countries, we continuously investigate to optimize the condition for multiplying FECs from Asian elite cultivars.Transformation technology for several industry-preferred African cultivars of cassava has been developed, however, few reproducible transformation technology has been developed for industry-preferred Asian cultivars of cassava, because the frequency of FEC induction is quite low and a protocol for the reproducible FEC production has not been established in most Asian cultivars. Although the transformation via SE for Asian cultivars has already been reported, the transgenic lines obtained were likely genetically unstable plants and/or chimeric transgenic plants . As nextS1 FigThe present study was modified on the culture conditions in step 5.(TIF)Click here for additional data file.S2 FigThe concentration of ammonium (A), nitrogen (B), phosphate (C), potassium (D), boron (E), calcium (F), magnesium (G), sulfur (H), Cl (Cl), microelements (J), and other microelements (K and L) used in the various media. MS, DKW, GD SH, McCW, Chu and GB5 indicate Murashige and Skoog medium [g medium , Driver-g medium , Gresshog medium , Schenk g medium  McCown'sg medium , Chu Click here for additional data file.S3 FigPrincipal component analysis of the gene expression identified by microarray (A), Frequency histogram of gene expression in microarray. The X-axis shows the normalized gene expression level. The Y-axis shows the number of genes at a given expression level (B). Red and blue circles on (TIF)Click here for additional data file.S4 FigThe graph was generated for the GO terms within the category biological process by Parametric Analysis of Gene Set Enrichment (PAGE) in agriGO . A total(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.


CDKN1A might act as a molecular switch to control spatial interactions between regulatory elements.A regulatory program involving hundreds of genes is coordinated by p53 to prevent carcinogenesis in response to stress. Given the importance of chromatin loops in gene regulation, we investigated whether DNA interactions participate in the p53 stress response. To shed light on this issue, we measured the binding dynamics of cohesin in response to stress. We reveal that cohesin is remodeled at specific loci during the stress response and that its binding within genes negatively correlates with transcription. At p53 target genes, stress-induced eviction of cohesin from gene bodies is concomitant to spatial reorganization of loci through the disruption of functional chromatin loops. These findings demonstrate that chromatin loops can be remodeled upon stress and contribute to the p53-driven stress response. Additionally, we also propose a mechanism whereby transcription-coupled eviction of cohesin from  It requires the establishment of an intricate gene expression program coordinated by the tumor suppressor transcription factor, p53. In response to stress, p53 integrates signals and directs cell fate by regulating the expression of genes involved in major cellular pathways fication , 2. Depee.g. enhancers, silencers) to their target promoters [Pioneering work recently revealed that the human genome has a spatial organization shaped by chromatin loops –8. The fromoters , 5, 15. romoters –18. ConsCDKN1A. Together, these findings demonstrate that chromatin loops are remodeled during the cellular stress response and contribute to the p53 transcriptional program.Given the importance of the spatial organization of the genome in the regulation of transcription, we explored whether DNA interactions are remodeled in response to stress to help establish the p53 transcriptional stress response program. To shed light on this issue, we used a genome-wide approach to investigate the binding of the cohesin subunit Rad21 in response to stress. Here, we show that cohesin is remodeled at specific loci during stress response. Notably, we unveil that cohesin is evicted from p53 target gene bodies during stress-induced transcription. Furthermore, using high-resolution Circular Chromosome Conformation Capture coupled to deep sequencing (4C-seq), we reveal that cohesin’s eviction from p53 target gene bodies in response to stress coincides with the disruption of chromatin loops that shape the spatial architecture of these loci. Finally, we show that these stress-dependent chromatin loops are important because they contribute to gene regulation of Daunorubicin and Polyethylenimine (PEI) were obtained from Sigma.2-containing atmosphere in DMEM (Wisent) supplemented with 10% fetal bovine serum (Sigma), 0.2U/mL penicillin G, and 100 mg/mL streptomycin (Invitrogen). Unless specified otherwise, cells stress treatment consisted of 250 nM daunorubicin (Sigma) for 8 h. All results presented with SEM were obtained from independent biological triplicates. Statistics were calculated using Student’s t-test.HCT116 p53+/+ and HCT116 p53-/- cells  were cultured in a 5% COChIP assays were performed as described previously using the antibodies listed in Rad21 ChIP-seq was performed similarly to regular ChIP experiments except for the following steps. For each treatment conditions six immuno-precipitations were performed. Immuno-precipitations were performed using Dynabeads coupled to protein A (Life Technologies) without pre-clearing. The libraries for deep-sequencing were prepared from 10 ng ChIPed DNA using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) according to manufacturer’s protocol. Samples were sequenced paired-end at 50-bp read length on an Illumina HiSeq-2000. See supplementary mRNAs of p21, p21C and 36B4 were quantified by RT-qPCR and normalized to 36B4 expression level. Total RNA was extracted from cultured cells using GenElute (Sigma) and reverse transcribed using the M-MLV reverse transcriptase enzyme (Enzymatics) and random hexamers according to manufacturer’s protocols. The RT-qPCR primer sets are reported in Total RNA was extracted from HCT116 p53+/+ cells using GenElute (Sigma) and treated with DNaseI (Ambion). The libraries for deep-sequencing were prepared from 800 ng total RNA using Illumina’s TruSeq Stranded mRNA LT Kit according to manufacturer’s protocol. Samples were sequenced paired-end at 50-bp read length on an Illumina HiSeq-2000. See supplementary Cells were grown in a 150 mm petri dish to 50–70% confluence and were then crosslinked in 1.1% formaldehyde for 10 min then washed two times in cold PBS. Cell pellet was resuspended in 500 μl of lysis buffer  and incubated for 30 min at 4°C on a rotary wheel. Subsequently, lysate was sonicated to obtain DNA fragments of 500 bp and was then centrifugated at 14,000 rpm for 10 min at 4°C. Supernatant was recovered and a 3 μl aliquote was collected as 1% input. Next, 500 μl of phenol/chloroform and 200 μl of lysis solution were added to 300 μl of lysate, the solution was then vortexed and centifugated at 12,000 rpm for 5 min. The aqueous phase was collected and transferred to a Gel-Lock tube containing 500 μl of phenol/chloroform. The solution was then mixed by inversion and centrifugated at 14,000 rpm for 1 min. The aqueous phase was collected, transferred to a tube containing 100 μg of proteinase K, and then incubated over-night at 65°C. Parallel to this, 500 μl of TE as well as 100 μg of proteinase K were added to the input aliquote, which was then incubated over-night at 65°C. The next day, 50 μg of RNase A were added to the samples, which were then incubated for 1 h at 37°C. DNA was then purified using QIAquick PCR Purification Kit and eluted using 700 μl of elution buffer. qPCR were subsequently performed using the primers reported in the 4C experiments were performed and analyzed as previously described , 21. ResWe used pGL3 basic, pGL3 promoter, and pRL-SV40 (Promega) to assess promoter, enhancer and repressor activity of putative regulatory elements. Cells were cultured in six-well plates, transfected using PEI with 100 ng of pGL3 vector, 2.5 ng of pRL-SV40, and 1000 ng of pGEX as carrier DNA, and collected 24h later. When a daunorubicin treatment was performed, HCT116 cells were transfected and treated 16 h later with 250 nM daunorubicin for 8h. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System kit from Promega and a Lumat LB 9507 luminometer from Berthold Technologies. Firefly signal was normalized to renilla signal. The sequences cloned in pGL3 vectors are reported in the 5’-ACACGACGrCrUrCrUrUrCrCrGrArUrCrU-3’ was then ligated to the mRNA. A 19 μl reaction containing 1 μg of purified RNA, 40 pmoles of 5’ adaptor, 1X T4 RNA ligase 1 buffer (New England Biolabs), 10% DMSO was assembled, incubated for 1 min at 65°C and then placed on ice. The reaction was then completed by adding 1 mM ATP, 20 u of RNasin (Promega), 30 u of T4 RNA ligase 1 (New England Biolabs) for a final volume of 25 μl and was incubated for 1 h at 37°C. RNA was then purified using SPRI agencourt RNAclean XP according to manufacturer’s instructions using a volume ratio of 1.8. A reverse transcription was then performed at 45°C using M-MLV reverse transcriptase enzyme (Enzymatics) according to manufacturer’s instructions using a gene specific primer 1 (GSP1)  and treated with DNaseI (Ambion). 1μg of total mRNA was then treated for 1 h with Tobacco Acid Pyrophosphatase (Epicentre) according to manufacturer's instructions and then purified using RNA Clean & Concentrator-5 (Zymo Research Corporation). The 5’ adaptor SP1) see . A PCR wimer see . PCR proet al. who showed that cohesin is evicted from the p53 target gene CDKN1A following 5-fluorouracil treatment, supporting that cohesin could be remodeled from its binding sites in response to stress [i.e. chromatin loops might be disrupted when cohesin is evicted from its binding sites. Hence, we used the cohesin subunit Rad21 as a proxy for cohesin and performed a ChIP-seq of Rad21 in HCT116 p53+/+ cells treated with the DNA damaging agent daunorubicin. RNA-seq experiments were carried out in parallel to obtain the transcription level of genes following stress induction. The biological replicates of these experiments exhibited a high degree of correlation  of Rad21 in HCT116 p53+/+ and HCT116 p53-/- cells treated or not with daunorubicin. We measured the binding of Rad21 at the CDKN1A (p21) site because CDKN1A induction in response to daunorubicin is virtually abolished in HCT116 p53-/- cells  [CDKN1A locus in response to stress: 1) the intragenic cohesin site, 2) the CDKN1A promoter, and 3) the NDR located downstream from the cohesin site  . Converste (TSS) –28. Nextte (TSS) . FAIRE rte (TSS) . We thussin site .CDKN1A locus in response to stress using high-resolution 4C   in HCT116 cells treated with daunorubicin. CDKN1A NDR and the 589–989 bp DNA fragment. We next investigated the nature of the transcript starting from this internal promoter. Nozell and Chen previously reported that p21C, a p21 variant, is transcribed from intron 1 of CDKN1A. p21C differs from p21 mRNA by its first non-coding exon that splices with the exon 2 of p21 [CDKN1A NDR, we speculated that the internal promoter might be responsible for the expression of p21C. Therefore, we determined the 3’ splice junction between the 5’ RACE product and the exon 2 of p21. A PCR using a forward primer overlapping the 5’ RACE product and a reverse primer located within exon 2 of p21 confirmed that the 5’ RACE product is spliced with exon 2 of p21  [Our data reveal that specific loci exhibit profound alteration in Rad21 binding following stress treatment. This happens equally at sites located outside genes or within introns and exons. However, we show that for Rad21 sites located within genes there is a correlation between the binding of Rad21 and the transcriptional status of the gene. For genes induced in response to stress, Rad21 binding decreases as gene expression increases, which is noticeable at p53 target genes . In the  cohesin . At leasg. Wapl) . Howeverg. Wapl) . Rather,g. Wapl) , 42. ThiCDKN1A and FDXR using high-resolution 4C reveals a correlation between cohesin eviction and the disruption of chromatin loops. We show that in non-stressed cells the CDKN1A locus is shaped by pre-established chromatin loops: a large interacting hub connects the CDKN1A promoter, PANDAR, and a cluster of three p53 binding sites, while the CDKN1A alternative internal promoter for p21C interacts solely with a downstream repressor element  Correlation between Rad21 ChIP-seq biological replicates. (B) Correlation between RNA-seq biological replicates. (C) Distribution of Rad21 ChIP-seq peaks with a Z-score <-2 and >2 within the genome. (D) RNA-seq and Rad21 ChIP-seq tracks obtained in HCT116 p53+/+ non-treated (NT) or treated with daunorubicin (Dauno) for the FDXR locus. (E) ChIP-seq signal of Rad21 binding sites surrounding the CDKN1A and FDXR genes. When Rad21 sites are located within genes, the transcription level fold induction of the gene following daunorubicin is indicated. (F) Correlation of Rad21 binding signal fold change with gene transcription fold change for Rad21 binding sites located within genes. (G) Correlation of Rad21 peak height and gene transcription FPKM for Rad21 binding sites located within genes. Data obtained in daunorubicin treated HCT116 cells. (H) p21 mRNA level assayed by RT-qPCR in HCT116 p53 +/+ and p53 -/- cells treated or not with daunorubicin.((DOC)Click here for additional data file.S2 FigA) mRNA induction in response to stress measured by RNA-seq for the p53 target genes FDXR, TP53i3, and GDF15 (B) ChIP of Rad21 and Smc1A at cohesin binding sites located within gene bodies of FDXR, TP53i3, and GDF15 in HCT116 p53+/+. (C) ChIP of Rad21 and Smc1A at cohesin binding sites located within gene bodies of FDXR, TP53i3, and GDF15 in HCT116 p53-/-.((DOC)Click here for additional data file.S3 FigA) The CTCF-Cohesin site present within the first intro of CDKN1A is conserved between different cell types. CTCF and Rad21 ChIP-seq signal at the CDKN1A gene is shown for different cell types. Representation adapted from the UCSC genome browser using tracks from the ENCODE project. (B) ChIP of CTCF and Rad21 performed in HCT116 p53+/+ cells. The signal obtained at the CDKN1A cohesin site is shown. (C) FAIRE experiment and ChIP experiment of H3K4me1, H3K4me3, and H3 carried out in non-treated HCT116 cells. H3K4me1 and H3K4me3 ChIP results were normalized to H3 to account for nucleosome density. (D) CDKN1A gene with the nucleosome depleted region shown in red. The DNaseI 125 cell types track , and the ChIP-seq track for Rad21 obtained in HCT116 p53+/+ non-treated are also shown. Adapted from the UCSC genome browser.((DOC)Click here for additional data file.S4 FigA) Representation of the CDKN1A gene with the promoter, the cohesin, and the NDR 4C viewpoints. 4C DpnII restriction sites are shown in red. The ENCODE DNaseI tracks  adapted from the UCSC genome browser is also shown. (B) Biological and technical replicate of the high-resolution 4C experiment carried out in HCT116 cells treated or not with daunorubicin using DpnII as first cutter. The NDR viewpoint track is shown. (C) High-resolution 4C experiment performed using Csp6I as first cutter (instead of DpnII) in HCT116 cells treated or not daunorubicin. The NDR viewpoint track is shown. (D) High-resolution 4C experiment carried out in HCT116 p53-/- cells treated or not with daunorubicin using DpnII as first cutter. The NDR viewpoint track is shown.((DOC)Click here for additional data file.S5 FigCDKN1A locus. HCT116 p53+/+ cells were treated (Dauno) or not (NT) with daunorubicin. The 4C tracks for the cohesin site and the interacting region are presented with their respective viewpoints in red. Rad21 ChIP-seq tracks are also shown. P53 binding sites are shown in green in the gene track.4C high-resolution interaction map of the (DOC)Click here for additional data file.S6 FigA) Luciferase reporter assay to test if the internal promoter activity is affected by stress. (B) Luciferase reporter assay to test if the activity of the internal promoter is p53-dependent. (C) PCR products obtained with primers amplifying the 5’RACE / p21 exon 2 junction using gDNA as template or cDNA from daunorubicin treated HCT116 cell.((DOC)Click here for additional data file.S7 FigA) 4C track of the NDR viewpoint obtained in non-treated HCT116 cells aligned to the ENCODE DNaseI track . Adapted from the UCSC genome browser. (B) FAIRE experiment carried out in non-treated HCT116 cells. Primer sets used were tailing the DNaseI peak located at chr6:36,660,086–36,660,295 strand (+) hg19. (C) Luciferase reporter assay to test the distal enhancer activity of the interacting region regulatory element. The signal is normalized to the Internal promoter/Luciferase construction. (D) Luciferase reporter assay to test the proximal enhancer activity of the interacting region regulatory element. The signal is normalized to the Control DNA1/Internal promoter/Luciferase construction. (E) Luciferase reporter assay to test the promoter activity of the interacting region regulatory element. The signal is normalized to the Control DNA1/Luciferase construction. (F) Luciferase reporter assay to test if the interacting region regulatory element represses SV40 enhancer activity. The signal is normalized to the SV40 promoter/Luciferase construction.((DOC)Click here for additional data file.S8 FigVenn diagram of the intersection of p53 binding sites and Rad21 binding sites in HCT116 p53+/+.(DOC)Click here for additional data file.S1 File(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 Table(DOC)Click here for additional data file.S8 Table(DOC)Click here for additional data file.S9 Table(DOC)Click here for additional data file.


Drosophila, the conserved Male-specific lethal (MSL) histone acetyltransferase complex mediates dosage compensation by increasing transcript levels from genes on the single male X-chromosome approximately two-fold. Consistent with its increased levels of transcription, the male X-chromosome has enhanced chromatin accessibility, distinguishing it from the autosomes. Here, we demonstrate that the non-sex-specific CLAMP (Chromatin-linked adaptor for MSL proteins) zinc finger protein that recognizes GA-rich sequences genome-wide promotes the specialized chromatin environment on the male X-chromosome and can act over long genomic distances (~14 kb). Although MSL complex is required for increasing transcript levels of X-linked genes, it is not required for enhancing global male X-chromosome chromatin accessibility, and instead works cooperatively with CLAMP to facilitate an accessible chromatin configuration at its sites of highest occupancy. Furthermore, CLAMP regulates chromatin structure at strong MSL complex binding sites through promoting recruitment of the Nucleosome Remodeling Factor (NURF) complex. In contrast to the X-chromosome, CLAMP regulates chromatin and gene expression on autosomes through a distinct mechanism that does not involve NURF recruitment. Overall, our results support a model where synergy between a non-sex-specific transcription factor (CLAMP) and a sex-specific cofactor (MSL) creates a specialized chromatin domain on the male X-chromosome.The essential process of dosage compensation is required to equalize gene expression of X-chromosome genes between males (XY) and females (XX). In  D. melanogaster and across Diptera, dosage compensation occurs by increasing transcript levels of thousands of genes along the length of the single male X-chromosome two-fold to equalize transcript levels with XX females [Dosage compensation is an ancient mechanism that functions to regulate transcription of X-chromosome genes to equalize transcript levels between XY males and XX females. There is mounting evidence that one conserved mechanism to equalize gene dosage is upregulation of the X-chromosome . In fact females –4. This  females .It was long thought that the MSL complex promotes an open chromatin environment on the X-chromosome through the H4K16 acetyltransferase activity of its Males absent on the First (MOF) component in order to facilitate increased transcription . The acecis-elements located within CES, both in the presence and absence of MSL complex [cis-elements are more clustered within CES than anywhere else in the genome [cis-elements elevate CLAMP occupancy on the X-chromosome compared with autosomes [We previously discovered that a non-sex-specific transcription factor, which we named Chromatin-linked adapter for MSL Proteins (CLAMP), is required for MSL complex recruitment to the male X-chromosome ,10. CLAM complex ,11. On tutosomes ,11. MSL utosomes .clamp null mutant larvae exhibit disrupted chromatin structure [In both males and females, CLAMP binds more frequently within gene bodies on the X-chromosome than autosomes  because tructure , suggestHere, we use genomic approaches to demonstrate that CLAMP can act over substantial genomic distances (~14 kb) to promote the formation of a broad domain of enhanced chromatin accessibility on the male X-chromosome. In contrast, MSL complex functions to increase chromatin accessibility specifically at CES peak centers, which are its sites of highest occupancy. Interestingly, CLAMP modulates chromatin accessibility over significant distances beyond the center of CES independent of MSL complex. At a subset of CES where CLAMP and MSL complex promote each other’s occupancy , CLAMP iTo examine whether CLAMP regulates chromatin accessibility, we identified nuclease-accessible regions genome-wide using a recently developed assay that is based on chromatin digestion in a MNase titration series followed by high-throughput sequencing  Fig 1A)Fig 1A). Drosophila male (S2) and female (Kc) tissue culture cells. S2 and Kc tissue culture cells are often compared to study Drosophila dosage compensation [clamp RNAi but not msl2 RNAi in Kc cells because MSL complex is not present in these female cells. As a control, we used a gfp RNAi treatment that activates the RNAi pathway but does not target any gene in the Drosophila genome [To test our hypothesis that CLAMP promotes global opening of the chromatin on the male X-chromosome, we performed MNase-seq titrations in ensation ,21 becauensation ,20,22. Fa genome .clamp RNAi efficiency using immuno-blotting and determined that no detectable CLAMP protein remains after clamp RNAi as we have previously reported  scores within an experiment to compare cell types and conditions on a global level . Additiomsl2 RNAi had no effect on the average chromatin accessibility scores of the X-chromosome or autosomes in agreement with a recent study [msl2 RNAi). In contrast, clamp RNAi reduced accessibility scores of the male X-chromosome to levels below that of autosomes (clamp RNAi). In females, clamp RNAi had no average effect on autosomes or the X-chromosomes relative to control RNAi (clamp or msl2 depleted samples. This approach showed that clamp RNAi treatment resulted in significantly more genomic locations (bins) with decreased accessibility than an increased accessibility on the male X-chromosome compared with the autosomes  changes in accessibility based on their location: within gene bodies, at transcription start or termination sites (TSS/TTS), at enhancers, or in unannotated regions (see clamp and msl2 RNAi treatments occur within gene bodies (>65% for both treatments), consistent with the most frequent genomic location of CLAMP and MSL complex binding sites  At Gclamp and msl2 RNAi reduced chromatin accessibility at Group A CES most strongly followed by Group B CES , clamp RNAi reduced chromatin accessibility outside of the CES peak but not at the peak . In contrast, clamp RNAi resulted in a broad loss of chromatin accessibility over the majority (86%) of male X-linked genes (clamp RNAi), while male autosomes and female chromosomes exhibited nearly equal percentages of genes with increased vs. decreased accessibility . These heat maps were rank-ordered based on the magnitude of accessibility change over the gene body following either reatment . We alsosibility .clamp RNAi over gene bodies after separating genes based on the enrichment of CLAMP within their TSS region. We determined groups by ranking CLAMP ChIP-seq enrichment from highest to lowest and dividing genes into two groups, “high” and “low”  in their Start-RNA abundance in male cells, while approximately 7% were significantly changed in female cells .To analyze the relationship between transcriptional and chromatin changes upon CLAMP depletion, we used the Start-seq technique . The Staclamp RNAi. We found that the significantly changed Start-RNAs were evenly distributed amongst all chromosomes in females, and modestly biased towards the X-chromosome in male cells, suggesting a role for CLAMP in transcriptional regulation on all chromosomes  changed Start-RNAs after omosomes . For eacamp RNAi .clamp RNAi are due to changes in accessibility or vice versa. However, in the case of the male X-chromosome, it is likely that changes in chromatin accessibility are separable from the transcriptional changes associated with MSL-dependent dosage compensation even if they are not separable from other transcription changes for the following reason: While MSL complex mediates the transcriptional changes associated with dosage compensation [The male X-chromosome had the greatest percentage of transcripts positively regulated by CLAMP (62%), which was expected given the role of CLAMP in male X-chromosome dosage compensation which increases transcript levels . While tensation ,22, it densation . Furtherensation . Therefoth chromosome   for the core Histone 3 (H3). We first measured the effect of gulated) . We obseromoters . This suclamp RNAi result from changes in nucleosome occupancy. We selected CES that exhibit CLAMP-dependent decreases in chromatin accessibility   . Thus, CRNA on the X (roX) RNAs in females [To define a possible mechanism by which CLAMP decreases histone occupancy at CES, we measured how CLAMP regulates recruitment of the Nucleosome Remodeling Factor (NURF) chromatin-remodeling complex. Several lines of evidence suggest a possible functional link between CLAMP and NURF. First, NURF has been previously implicated in regulating the chromatin organization of the male X-chromosome . Second, females ,34. Thir females ,36. Four females –41. Therclamp RNAi treatment. Co-occupancy of CLAMP and NURF301 was confirmed at each of these sites using previously published CLAMP ChIP-seq and NURF301 ChIP-chip data from the modENCODE project , a GA-repeat zinc finger protein with a similar domain structure and sequence recognition element to CLAMP [Interestingly, we found that CLAMP does not regulate accessibility at all CES similarly. For example, while CLAMP promotes chromatin accessibility at Group A CES, to CLAMP . Like CLto CLAMP –41. We hto CLAMP , indicatCLAMP is a zinc finger protein that promotes the recruitment of different effector complexes that regulate chromatin accessibility including the MSL complex and NURF. Previous work has implicated the NURF chromatin remodeler complex in regulating the chromatin organization of the male X-chromosome . Indeed,et al., submitted). In this way, CLAMP-mediated changes in accessibility at TSS could be mediated by transcriptional regulation through a functional association between CLAMP and NELF. Future work will be required to examine the link between CLAMP and NELF occupancy genome-wide to determine if and how this relationship facilitates chromatin accessibility at transcription start sites.In addition to regulating chromatin accessibility of the X-chromosome, CLAMP alters chromatin accessibility at TSS throughout the genome in both males and females. However, this occurs independently of NURF or changing histone occupancy levels at a subset of sites tested . At promWhile many transcription factors bind to thousands of locations throughout the genome, they often function differently depending on their genomic contexts –50. For cis-elements that target CLAMP became enriched on the X-chromosome due to the activity of transposons [We suggest that CLAMP functions as an adapter protein that associates with different complexes at distinct genomic locations to perform diverse context-specific functions. Because CLAMP is a highly conserved  and matensposons , MSL comDrosophila S2 and Kc cells were maintained at 25°C in Gibco Schnieder’s Drosophila media (ThermoFisher Scientific) supplemented with 10% heat-inactivated Fetal Bovine Serum and 1.4X Antibiotic-Antimycotic (ThermoFisher Scientific). Cells were passaged every 2–3 days to maintain appropriate cell density.gfp (control), clamp, and msl2 for RNAi has been previously validated and described [clamp and msl2 dsRNA were generated using PCR amplification from bacterial artificial chromosomes (BAC) available from the BACPAC resources program  [Generation of dsRNA targeting escribed ,20,57. I1 59O03) . PCR pro7 S2 or Kc cells suspended in 6mLs of Gibco Schenieder’s Drosophila media without FBS were added to a T75 tissue culture flask containing 135μg gfp, clamp, or msl2 dsRNA in 3mL ultra pure water. The cells were serum starved at room temperature for 45 minutes before adding 6mLs of Gibco Schneider’s Drosophila media supplemented with 10% FBS to the flask containing the dsRNA and cells. Cells were incubated for 6 days and upon collection efficiency of the RNAi treatment was determined before performing the desired experiment.RNAi was performed in T75 tissue culture flasks. A total of 1.2x10Following 6 days of RNAi treatment, cells were scraped and 500μL was collected for immuno-blotting. To extract protein, cells were first pelleted at 5,000xg for 3 minutes at 4°C. Cell pellets were next washed in 100μL 1X phosphate buffered saline before a second centrifugation. The supernatant was removed and the cell pellets were resuspended in 40μL of lysis buffer ). After a 5-minute incubation at room temperature, the lysates were vortexed briefly. The samples were then cleared by centrifugation at room temperature at 14,000xg for 10 minutes. The supernatant was transferred to a new tube and the protein abundance was quantified using a Qubit Fluorometer (ThermoFisher Scientific).To immuno-blot, a total of 5μg of protein was loaded on a pre-cast Tris Glycine gel (ThermoFisher Scientific) and immobilized on PVDF membrane using the iBlot transfer system (ThermoFisher Scientific). CLAMP  and Actin  proteins were detected using the Western Breeze kit (ThermoFisher Scientific) following the manufacturer’s instructions.msl2 and roX2 following RNAi treatment, 500μL of cells were collected following the 6-day incubation period. After pelleting cells and removing the media, total RNA was extracted using the RNeasy Plus RNA extraction kit (Qiagen). A total of 1μg of RNA was reverse-transcribed to cDNA using the SuperScript Vilo cDNA Synthesis kit (ThermoFisher Scientific) by following the manufacturer’s instructions. Targets were amplified from cDNA using validated primers at a concentration of 200nM. Primer sequences for qRT-PCR are available upon request. Three technical replicates for each sample were amplified using SYBR Green on an Applied Biosystems StepOnePlus™ Real-Time PCR System. The obtained relative abundance values were averaged and used to calculate ΔCt relative to PKA as an internal control.To determine transcript abundance of Drosophila S2 and Kc cells for RNAi treatment with gfp, clamp, or msl2 was performed in T75 flasks. MNase titration was performed after 6 days of RNAi treatment as previously described [Preparation of two biological replicates of escribed .gfp (control) or clamp dsRNA. RNAi for Start-seq was performed as described above for MNase-seq with the exception that T150 flasks were used instead of T75. This resulted in a doubling of all reagents to perform the RNAi treatment. Total RNA was extracted from nuclei using Trizol reagent (Invitrogen) and transcription start site RNAs were isolated and prepared for sequencing as previously described [Start-sequencing was performed from three biological replicates of S2 and Kc cells that were treated for 6 days with either escribed . Analysiescribed .gfp (control) and clamp dsRNA treated cells prepared in T150 flasks after 6 days of incubation. To prepare for chromatin immunoprecipitaton, cells were first scraped from the bottom of the flask to resuspend adhered cells. Formaldehyde (37% stock) was added to a final concentration of 1% and the cells were crosslinked for 10 minutes by shaking at room temperature at 110 rpm. The crosslinking reaction was quenched with the addition of 2.5M glycine to a final concentration of 125mM. Fixed cells were transferred to a 15mL falcon tube and placed on ice.Chromatin was prepared from Following crosslinking and quenching, the media containing formaldehyde and glycine was removed after pelleting cells by centrifugation at 4°C for 5 minutes at 1,500xg. The cells were washed briefly by resuspending in 15mL of PBS-EDTA . Cells were pelleted as before and the supernatant removed. Next, the cells were resuspended in 6mL of ChIP wash A  and rotated for 10 minutes at 4°C. After rotation, the cells were pelleted once again and the supernatant removed. The cell pellet was then resuspended in 6mL ChIP wash B . The cells were rotated for 5 minutes at 4°C. A small aliquot was removed to determine total cell quantity using Trypan Blue (ThermoFisher Scientific). After washing the cells in ChIP wash B, the cells were pelleted flash frozen in liquid nitrogen for storage at -80°C.8 cells/mL. The cells were lysed for 10 minutes by rotating at 4°C before aliquoting equal volumes (between 100-300uL) into Protein LoBind Safelock tubes (Eppendorf). The cell lysates were then sonicated for 3 cycles totaling 5 minutes each on high setting using a water bath sonicator  programmed to pulse 30 sec on, 30 sec off. After sonication, replicate samples were pooled and centrifuged at 4°C for 10 minutes at a speed of 13,000 rpm to pellet the insoluble chromatin. Solubilized chromatin was transferred to a 15mL falcon tube, to which 9 volumes of ChIP dilution buffer was added . Diluted chromatin was filtered on a PolyPrep chromatography column (BioRad) before aliquoting 1mL into Protein LoBind safelock tubes. Additionally, 400uL was removed for Input DNA. Chromatin aliquots were flash frozen for storage at -80°C until after Input was checked for chromatin size distribution.Using the total cell count, cell pellets were resuspended in an appropriate amount of lysis buffer  to obtain a cell concentration of 1x10In order to determine the size distribution of chromatin fragments, the 400uL of Input DNA was reverse crosslinked by incubation overnight at 65°C after the addition of 21.5uL 20% SDS , 15uL of 5M NaCl  and 1uL of RNase A (Roche). Next, an additional 1uL of RNase A was added for 30 minutes at an incubation temperature of 37°C. After this, protein was digested from the chromatin by adding 20uL of 1M Tris-HCL pH 6.8 , 10uL EDTA pH 8.0 , and 3uL 20mg/mL Proteinase K  and incubating for 90 minutes at 45°C. Following protein digestion, a standard phenol-chloroform extraction was performed followed by ethanol precipitation. DNA pellets were resuspended in 100uL ultra pure water and quantified. To determine fragmentation distribution was between 100-500bp, 200ng of chromatin was visualized on a 2% agarose gel.To begin the immunprecipitation, either 2.5ug of NURF-301 antibody  or 2ug of Histone H3 (Abcam #ab1791) was added to 1mL of previously prepared chromatin and rotated overnight at 4°C. The next day, 60uL of salmon sperm blocked Protein A agarose beads (Millipore) were added and the samples rotated for an additional 2 hours at 4_C. After incubation, the beads were pelleted by centrifugation at 4°C for 3 minutes at 1,000xg. The beads were washed a total of 6 times in a series of wash solutions. For each wash, first the beads were resuspended in 1mL solution, then rotated for 5 minutes at 4°C. Beads were then pelleted by centrifugation at 1,000xg for 3 minutes at 4°C, the supernatant was removed, and the beads resuspended in the next wash solution. The washes include: 1.) two washes in RIPA 150 Buffer ; 2.) one wash in RIPA 300 buffer , 3.) one wash in LiCl/TE buffer , and 4.) two washes in TE buffer . After the final TE buffer wash, the beads were eluted twice in 250uL sodium bicarbonate Elution Buffer  by rotating at room temperature for 15 minutes and pelleting by centrifugation at room temperature for 3 minutes at 1,000xg to collect the eluate. The 500uL of immunoprecipitated samples were reversecrosslinked overnight at 65°C after the addition of 20uL 5M NaCl . After reverse crosslinking the immunoprecipitated samples overnight, 1uL of RNase A (Roche) was added and the samples incubated for 30 minutes at 37°C. Next, 20uL 1M Tris-HCL pH 6.8 , 10uL 0.5M EDTA pH 8.0 , and 3uL 20mg/mL Proteinase K were added to the samples and incubated for 90 minutes at 42°C. After digesting proteins, the DNA from the immunprecipiated samples was cleaned by using a standard phenol:chloroform extraction followed by ethanol precipitation. The immunoprecipitated DNA pellet was then resuspended in 100uL ultra pure water for downstream analysis by qRT-PCR.™ Real-Time PCR System. The obtained relative abundance values were then averaged and used for calculation of enrichment.To determine enrichment of NURF301 or H3 to chromatin entry sites and promoters, three independent biological ChIP replicates were performed. Targets were amplified from 2uL of Input (1%) and immunoprecipitated DNA using validated primers at a concentration of 200nM. Primer sequences for qRT-PCR are in cg15570 control gene, which is a genomic locus unbound by CLAMP as determined by ChIP-seq. Internally normalized values were then normalized to the 1% Input sample to determine log2-fold enrichment of the immunoprecipitated sample compared to Input. Subsequently, the enrichment values from the three biological replicates were averaged and standard error of the mean was calculated.Internal normalization was performed using primers located within the CLAMP ChIP-seq data and MSL CES coordinates were taken from GSE39271. To determine overlap between CLAMP and NURF301, browser tracks were taken from GSE20829 (S2 cells) and GSE32845 (Kc cells). Data from this study is deposited to NCBI GEO with GEO number GSE99894.D. melanogaster reference genome (dm3) using Bowtie aligner [Z-score = 7) were identified, and the tags mapped to such positions were discarded [clamp RNAi treatment is reproduced when MACC values were median-shifted to zero for each sample independently . Significance of accessibility or transcription change was calculated in R using the ‘limma’ package. Mann-Whitney test was used to estimate the significance of the observed effects.Gene coordinates were taken according to the dm3 annotation. The modENCODE annotation of the enhancers that are active in S2 cells were used in the analysis . TSS . C) Transcript abundance of roX2 following msl2 RNAi was measured as a functional test for efficiency of the RNAi treatment. Following msl2 RNAi treatment, roX2 abundance was significantly reduced in male cells, indicating functional reduction of MSL complex.A) An anti-CLAMP and anti-Actin western blot was performed to confirm efficient reduction of CLAMP (62 kDa). Actin (42 kDa) is used as a loading control. B) Transcript abundance of (PDF)Click here for additional data file.S2 Figmsl2 RNAi in males compared to control. In both males and females, treatment with clamp RNAi results in an overall decrease in MACC values. For all box and whisker plots, the median MACC value is plotted with the notch at the median line representing the 95% confidence interval. B) The distribution of MACC values from all experimental conditions is shown as in A for each of the replicates separately. MACC scores from the replicates are in strong agreement. C and D) The overall distribution of MACC scores is shown separately for each replicate for the X-chromosome (blue) and autosomes (red) of Control (gfp), clamp and msl2 RNAi treated male (C) and female (D) cells. E) The difference in MACC value (Δ MACC) between control and RNAi treatment for an individual locus on either the X-chromosome or autosomes was calculated for both replicates separately. In males, the change in MACC scores indicates a reduction in X-chromosome accessibility following clamp RNAi but not msl2 RNAi. F) Analysis of the per-bin pair-wise difference in the chromatin accessibility (MACC) between RNAi conditions. The computed MACC valued were additionally median-shifted to zero in each sample independently prior to cross-sample comparison. G) To compensate for different number of X and autosomes, the reads that aligned to the X-chromosome were counted twice. The MACC values were additionally median-shifted to zero as in (F).A) The distribution of MACC values from all experimental conditions shows no effect on overall MACC scores after (PDF)Click here for additional data file.S3 Figclamp (green), and msl2 (purple) RNAi are plotted centered on PionX sites.A) Accessibility changes for different classes of genomic regions were normalized by the percentage of the genome covered by each feature. A change in accessibility was classified as either within a gene body (blue), at TSS/TTS (red), at an enhancer (green), or unannotated (purple). B) CLAMP ChIP-seq peaks  were sep(PDF)Click here for additional data file.S4 Figclamp or msl2 RNAi in male (S2) cells, and clamp RNAi in female (Kc) cells. Below, the difference in accessibility between control and RNAi treatment on the X-chromosome and autosomes is shown in the second row. Each heat map is rank-ordered by the level of CLAMP enrichment from ChIP-seq occupancy (shown on the left in green). B) Average MACC profiles along gene bodies are shown for male and female cells separated into X-chromosome and autosome plots. Shown are MACC profiles for genes that are lowly enriched for CLAMP with clamp RNAi treatment in green and control in blue. The dark line represents the average MACC value, while 95% confidence intervals are represented by the lighter colors.A) Heatmaps of MACC scores over gene bodies are shown for all annotated genes upon control, (PDF)Click here for additional data file.S5 Figclamp (green) and control (blue) is indicated, where the dark line represents the average MACC value, while 95% confidence intervals are represented by the lighter colors. B) The distribution of average MACC values around randomized TSS in non-repetitive regions is plotted for male (S2) cells. The darker line represents the average MACC value and the lighter shading indicates the 95% confidence interval. C and D) The nucleosome read counts obtained for each concentration of MNase are shown under control (blue) and clamp (green) RNAi conditions and centered over annotated TSS. Each concentration is shown as a gradient color of the RNAi treatment. Nucleosome profiles are shown for the male X-chromosome and autosomes separately for both males (C) and females (D). E) Average MACC values (darker line) were plotted +/- 500bp centered on transcription termination sites (TTS) separated by the X-chromosome and autosomes in males and females. There is a reduction in accessibility after clamp RNAi (green) compared to the control (blue). The lighter shading surrounding the mean line on all plots represents the 95% confidence interval. F) The percentages of significantly changed transcripts (p<0.05) in males (left) and females (right) are shown for each chromosomal arm. The blue bar indicates the X-chromosome and autosomes are in red/pink. G) The percentages of significantly changed transcripts (p<0.05) that decrease in abundance after clamp RNAi (un-hatched) or increase in abundance after clamp RNAi (hatched) are shown for each chromosomal arm in males (left) and females (right).A) Average MACC profiles for genes with low enrichment of CLAMP are shown centered on transcription start sites (TSS) and separated into X-chromosome and autosome plots for male and female cells. RNAi treatment of (PDF)Click here for additional data file.S6 FigProfiles were generated for both males (A) and females (B). X-chromosome and autosome obsTSS were categorized into quartiles based on the ability of CLAMP to positively or negatively regulate transcription as measured by Start-seq. Shown are the profiles for each quartile.(PDF)Click here for additional data file.S7 FigX-chromosome and autosome obsTSS were categorized into quartiles of increasing expression level as determined by transcript abundance in the control RNAi condition and separated by whether they are positively or negatively regulated by CLAMP.(PDF)Click here for additional data file.S8 FigX-chromosome and autosome obsTSS were categorized into quartiles of increasing expression level as determined by transcript abundance in the control RNAi condition and separated by whether they are positively or negatively regulated by CLAMP.(PDF)Click here for additional data file.S9 Figclamp, and msl2 RNAi treatment are shown in blue where dark blue indicates positive values and light blue are negative. The average number of sequencing reads from the four MNase-seq experiments generated a nucleosome profile that is shown in purple for Control, clamp, and msl2 RNAi. NURF301 recruitment was tested following clamp RNAi treatment by ChIP qRT-PCR at four promoters in males (A) and females (B), where the red bar underneath the gene is scaled to 100 bp. Similarly five CES were tested in males (C) and females (D). The black bar underneath the red CES bar indicates the location of the qRT-PCR product for the ChIP qPCR experiments.For each, enrichment for NURF301 (orange) and CLAMP (green) is shown. The MACC values after control, (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Thus, Identifying active cis-regulatory regions in the human genome is critical for understanding gene regulation and assessing the impact of genetic variation on phenotype. The developments of high-throughput sequencing and machine learning technologies make it possible to predict cis-regulatory regions genome wide.In the human genome, 98% of DNA sequences are non-protein-coding regions that were previously disregarded as junk DNA. In fact, non-coding regions host a variety of cis-regulatory regions. Using models for well-characterized cell lines, we identify key experimental features that contribute to the predictive performance. Applying DECRES, we delineate locations of 300,000 candidate enhancers genome wide , and 26,000 candidate promoters (0.6% of the genome).Based on rich data resources such as the Encyclopedia of DNA Elements (ENCODE) and the Functional Annotation of the Mammalian Genome (FANTOM) projects, we introduce DECRES based on supervised deep learning approaches for the identification of enhancer and promoter regions in the human genome. Due to their ability to discover patterns in large and complex data, the introduction of deep learning methods enables a significant advance in our knowledge of the genomic locations of cis-regulatory regions will provide broad utility for genome interpretation from functional genomics to clinical applications. The DECRES model demonstrates potentials of deep learning technologies when combined with high-throughput sequencing data, and inspires the development of other advanced neural network models for further improvement of genome annotations.The predicted annotations of The online version of this article (10.1186/s12859-018-2187-1) contains supplementary material, which is available to authorized users. CRRs play a crucial role in precise control of gene expression. Promoters and enhancers act via complex interactions across time and space in the nucleus to control when, where and at what magnitude genes are active. CRRs, through interactions with proteins such as histones and sequence-specific DNA-binding transcription factors (TFs), help specify the formation of diverse cell types and respond to changing physiological conditions. While gene expression is ultimately a reflection of regulation across multiple processes, the key role of promoters and enhancers has been a central focus of genome annotation for the past decade. The investment in generating informative data for the detection of these regions has been immense, in part motivated by the anticipation that advanced computational approaches would be able to transform the data into a reliable annotation of the genome.In this article, we apply deep supervised analysis methods to identify the positions of active Promoters and enhancers were early discoveries during the molecular characterization of genes. While promoters specify and enable the positioning of RNA polymerase machinery at transcription initiation sites, enhancers modulate the activity of promoters from linearly distal locations away from transcript initiation sites , 2. The The use of computational methods to detect the locations of promoters and enhancers has been a key focus of bioinformatics for twenty years . WitDespite the limited set of precisely annotated active enhancers, supervised machine learning models have been attempted to predict enhancer regions. In each case, a distinct definition of a suitable positive training set of enhancers was taken. A random-forest method was used in  to classNew laboratory methods are emerging, providing a refined resolution of CRR locations. The majority of human DNA is transcribed, producing diverse types of RNA. In particular, transcripts generated at the edges of enhancers, enhancer RNAs (eRNAs), allow for the experimental readout of active regulatory regions. Global run-on and sequencing (GRO-seq) protocols  measure We have previously proposed and herein present the use of a deep feature selection (DFS) model for the supervised prediction of CRRs . Deep leOur study stands on three important legs. First, the precisely annotated FANTOM promoters and enhancers, which provide the largest experimentally defined collection of CRRs. Second, the ENCODE project genome-wide feature data, such as histone modifications, TF binding, RNA transcripts, chromatin accessibility, and chromatin interactions. Third, deep learning methods to distinguish CRRs based on the available data. We unite the three components to create the DECRES (DEep learning for identifying Cis-Regulatory ElementS and other applications) model, with which we identify the most comprehensive collection of CRRs across the human genome yet compiled.We investigated the capacity of deep learning models to separate enhancers and promoters, and to distinguish them from other regions and between activity states. We trained a deep feedforward neural network over our balanced labelled training sets to predict our  test sets from each well-characterized cell type, repeating the procedure 100 times. The deep model takes experimentally derived features over genomic regions as inputs and outputs class labels of these regions with probabilities  Fig. a. We use11p = 5.57E-5 and 9.90E-5 for DECRES versus ChromHMM and Combined respectively, two tailed Student’s t-test; see Fig. 2p=7.36E-5 and 2.33E-4 for DECRES versus ChromHMM and Combined respectively, two tailed Student’s t-test; see Fig. 2To assess the relative utility of our supervised deep method for CRR prediction, we compared it with the unsupervised ChromHMM and ChromHMM-Segway Combined methods , 12 usin2bona fide regulatory regions.As the initial evaluation focused on FANTOM eRNA-based annotation of CRRs, the type of data used to train our supervised model, we sought to assess performance on data generated by alternative methods. We identified two independent collections of laboratory validated enhancers to further assess the performance of DECRES: a CRE-seq collection of regions tested in K562 cells  and MPRAp = 0.014, two-sided Mann-Whitney rank test).In the first independent set, subsets of predicted K562 enhancers and negative regions (as predicted by the Combined ChromHMM and Segway method) were assessed in the laboratory using CRE-seq . In thatp = 0.05, two-sided Mann-Whitney rank test). We used DECRES to predict the classes of the MPRA positive and MPRA negative enhancers. Our result in Additional file p= 4.8E-6 and p= 2.3E-6 for K562 and HepG2 respectively, two-sided Mann-Whitney rank test)  containing predicted TF binding sites for cell-selective TFs were tested using a massively parallel reporter assay (MPRA) . Only 41p=1.90E-08 and 5.06E-08 for enhancers and promoters respectively, two-tailed Student’s t-test; see Fig. 3p=1.86E-09, two-tailed Student’s t-test; see Fig. 3p=2.79E-01, 6.56E-01 and 1.17E-01 in Fig. Recent studies confirmed that DNA sequence properties can be useful for the recognition of promoters and enhancers , 5, 25, 3As experimental data can be time consuming and expensive to produce, we sought to determine the minimal set of features most informative for CRR prediction from a computational perspective. We used randomized deep feature selection (randomized DFS or RDFS) and random forest (RF) models (see Methods) for two-class [A-E+A-P (or CRR) versus BG] and three-class (A-E versus A-P versus BG) classifications on four cell types  which have 72-135 features available.Figure 4For the development of machine learning methods in genome annotation, minimizing the number of features required decreases cost and increases the capacity for biological interpretation. Figure 44The distributions of the top ten features for three-class predictions  are given in Additional file Additional file We trained 2- and 3-class multilayer perceptron (MLP) models (see Methods) using all reference (labelled) data for training, in order to predict CRRs across the entire genome for six cell types (A549 and MCF7 were excluded). The 2-class model identified 227,332 CRRs (adjacent regions were merged), which occupy 4.8% of the genome  enhancers will not have been detected in the original compilation of the enhancer atlas. Hence, we sought to identify additional partially supported enhancers for which eRNA signals were below the original atlas threshold settings . In the We performed functional enrichment analysis on the genome-wide predicted A-Es and A-Ps using GREAT . For GM1We further carried out motif enrichment analysis on the predicted cell-specific CRRs and not-in-atlas enhancers using HOMER . The preWe performed functional and enrichment analysis on the A-E and A-P predictions from the Combined method , and repcis-regulatory regions in the human genome would inspire the machine learning community to push the performance limit of supervised CRR-prediction methods, and would encourage laboratory biologists to accelerate cell type-specific data generation.Our study brings together a large collection of high-throughput data from global projects to allow for supervised annotation. One key challenge in such analysis is the depth of validation. In this report, validation is assessed using existing collections of reliable enhancers, including CAGE , and labEnhancers and promoters have both common and distinct characteristics. In our cross-validations, we show that A-E and A-P are highly separable Fig. a, while Our predicted CRRs take a substantial but small portion of the non-coding regions, previously known as “junk DNA”. It may be because only six cell types are considered in this work. Nevertheless, we have already seen that the non-coding regions exhibit regulatory functionality. It would be interesting in the next phase to collect data from a large number of cell types and examine the coverage, which will unveil whether regulatory regions have an oasis pattern. It may also imply that certain fragments of the non-coding regions play other partially known  and unknown roles.cis-regulatory elements of focus. Furthermore, transfer learning  in the training set, and applied the estimated scaling factors from the training set to the test set. From the most important to the least important features, we kept adding features to a MLP to evaluate how many features are sufficient for the prediction. Randomized DFS was compared to random forest on the same partitions of training and test sets in this study. The DFS and randomized DFS models were included in the DECRES package.The randomized DFS procedure is similar to random forest, both implementing the philosophy of the wisdom of crowds. In randomized DFS, DFS with perturbed feature-wise sparsity control parameters runs on a training subset which is a randomly sampled portion  using x and y axes, we qualitatively used t=0.5.Aiming at trading off the size of feature subset and classification performance (auPRC), we designed a method based on curving fitting that was applied in Fig. Additional file 1Tables S1-S7, and supplemental Figures S1-S30. (PDF 27600 kb)This file contains supplemental Additional file 2cis-regulatory regions for all six cell types. (ZIP 20400 kb)Genome-wide predictions of Additional file 3Predictions on CAGE supported bidirectional loci. AiA: Active in the FANTOM Enhancer Atlas; IiA: Inactive in the FANTOM Enhancer Atlas; NiA: Not included in the FANTOM Enhancer Atlas; Specific: Predicted cell-specific A-Es. (ZIP 4820 kb)Additional file 4List of features used for each cell type. (ZIP 1.36 kb)


Trypanosoma brucei, in which RNA polymerase II transcription initiation occurs over broad regions without distinct promoter motifs and lacks regulation. Using a combination of site‐specific and genome‐wide assays, we identified GT‐rich promoters that can drive transcription and promote the targeted deposition of the histone variant H2A.Z in a genomic context‐dependent manner. In addition, upon mapping nucleosome occupancy at high resolution, we find nucleosome positioning to correlate with RNA pol II enrichment and gene expression, pointing to a role in RNA maturation. Nucleosome positioning may thus represent a previously unrecognized layer of gene regulation in trypanosomes. Our findings show that even highly dispersed, unregulated promoters contain specific DNA elements that are able to induce transcription and changes in chromatin structure.Genome‐wide transcription studies are revealing an increasing number of “dispersed promoters” that, unlike “focused promoters”, lack well‐conserved sequence motifs and tight regulation. Dispersed promoters are nevertheless marked by well‐defined chromatin structures, suggesting that specific sequence elements must exist in these unregulated promoters. Here, we have analyzed regions of transcription initiation in the eukaryotic parasite  Two key features of this process are a core promoter, which functions as a platform on which the transcription machinery assembles, and a chromatin structure that allows the transcription machinery to access the promoter DNA  with no clustering of functionally related genes  than the remainder of the genome.We have generated for the first time genome‐wide maps of nucleosome occupancy and RNA pol II enrichment in Unexpectedly, we find RNA pol II levels and nucleosome occupancy to correlate not only at TSRs but also upstream of most genes, even within PTUs, pointing to a role for nucleosome positioning in RNA processing, rather than transcription initiation.T. brucei, RNA pol II transcription is thought to initiate within ~10‐kb‐wide regions enriched in H2A.Z . This required the identification of a non‐transcribed, yet transcriptionally permissive region of the genome. The organization of the T. brucei genome into long PTUs means that most of the core genome is actively transcribed, the exceptions being small regions between PTUs  upstream of a luciferase gene . When repeating the assay using the different TSR fragments, we observed that all 12 fragments were capable of initiating transcription well above background  these elements are enriched within TSRs, and (iii) they are unevenly distributed across the coding and non‐coding strands, giving rise to the observed bias in sense vs. antisense transcripts.Our analysis of primary transcripts indicated that transcription initiation has a strong strand bias. To determine whether TSR‐derived sequence elements are sufficient to ensure directionality , we inverted TSR fragments in the reporter assays  composed of 10mers that are enriched on the coding strand . Integraet al, To compare the activity of the GT‐rich sequence elements to that of endogenous RNA pol I and RNA pol II transcription, we targeted an rRNA promoter‐driven luciferase gene to the rDNA spacer and inserted a promoter‐less luciferase gene within a RNA pol II‐transcribed PTU. Not surprisingly, we find rRNA promoter‐driven luciferase expression to be 38.7‐ to 193.6‐fold higher when the transgene is located in the rDNA spacer compared to the region between two divergent RNA pol II TSRs. In agreement with earlier studies  occur at intervals of 10 bp. The exact positioning of the histone octamer with respect to the ~10‐bp helical repeat is termed “rotational positioning”  and depleted from long supra‐mononucleosomal fragments (> 175 nt) Fig A. To valThese findings indicate that DNA associated with H2A.Z‐containing nucleosomes is more easily digested than DNA bound to canonical nucleosomes, revealing TSRs as regions of increased DNA accessibility.Plasmodium falciparum  flanked by strongly positioned nucleosomes at the site of transcription initiation   tract, located just upstream of the SAS. At the RNA level, polyY tracts serve as binding sites for the U2AF65 subunit of the spliceosome  a polyY tract found upstream of the highly expressed GPEET procyclin genes, (ii) a long T‐rich polyY tract shown to be among the most efficient in transient trans‐splicing assays, or (iii) no polyY tract. All constructs contained an identical GPEET 5′ UTR sequence. Luciferase levels were highest for the GPEET polyY tract, twofold lower for the T‐rich polyY tract, and virtually absent in the cell line lacking a polyY tract . The additional nucleosome occupancy maps were interesting in several aspects. First, we noticed that in all three cell lines, the GT‐rich sequence was strongly depleted of nucleosomes Fig B. We susComparing the three additional nucleosome occupancy maps, it became apparent that the homopolymeric nature of the long T‐rich polyY tract, which should give rise to a rather rigid DNA double helix, resulted in an expansion of the NDR  or a derivative “single marker” cell line (SM) expressing T7 RNA polymerase and the Tet repressor  and RNeasy® MinElute® Cleanup (Qiagen) following the instructions of the manufacturer. Two micrograms of small RNA was treated with 1 unit of Terminator™ 5′‐Phosphate‐Dependent Exonuclease  in 1× Terminator Reaction Buffer B for 30 min at 42°C to remove 5′‐monophosphate RNA. Next, the RNA was purified and one half (+5′‐polyphosphatase) was treated with 20 units RNA 5′‐polyphosphatase to convert 5′‐triphosphate RNA to 5′‐monophosphate RNA. The second half (−5′‐polyphosphatase) was left untreated and served as a control. The sequencing libraries were constructed using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England BioLabs) and sequenced on an Illumina® NextSeq 500.Small RNA (< 200 nt) was purified from 5 × 10RPB9, a subunit of the RNA pol II complex, contained an endogenous Ty1 tag. Except for minor changes, the ChIP was performed as described previously  coupled to a BB2 antibody , and resuspended in 200 μL 1× PBS. In a 96‐well plate, 50 μl cell suspension (1.25 × 106 cells) and 50 μl Dual‐Glo® Luciferase Reagent were mixed, and after 10 min, firefly luciferase activity was measured for 1 s in a Victor Light Luminometer. Fifty microliters Dual‐Glo® Stop&Glo® Reagent was added, and after 10 min, Renilla luciferase activity was measured. All measurements were performed at least in duplicate. To normalize for differences in cell number, firefly luciferase activity was normalized with Renilla luciferase activity. All raw and normalized luciferase measurements are listed in Luciferase activities were measured with the Dual‐Gloet al, Polyclonal antibodies specific for H2A.Z were raised by immunizing rabbits with the following peptide: DDAVPQAPLVGGVAMSPEQAS, following a 145‐day immunization protocol (Pineda Antikörper‐Service). The antisera were affinity‐purified using SulfoLink Coupling Gel (ThermoFisher) with the immobilized peptide as described elsewhere  alleles was introduced by using the pyrFEKO system  coupled to polyclonal H3 rabbit antiserum . For visualization of the aligned reads, coverage values for the region of interest were extracted and visualized with GraphPad Prism version 5.0b.Immunoprecipitated DNA samples were sequenced in paired‐end mode using an Illumina HiSeq 2500 or an Illumina NextSeq 500 sequencer with 2 × 100 and 2 × 76 cycles, respectively. The processing of the sequencing data was performed as described previously . The computational data analysis was implemented as a Unix shell script, which together with further programs generated for this study is available at https://doi.org/10.5281/zenodo.438156.All sequencing data generated for this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar TNS designed research; CW and RD performed research; CW, KUF, and TNS analyzed data; and CW and TNS wrote the manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Dataset EV3Click here for additional data file.Dataset EV4Click here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Review Process FileClick here for additional data file.