Caenorhabditis elegans, the let-418 gene encodes one of the two Mi2 homologs, which is part of at least two chromatin complexes, namely the Nucleosome Remodeling and histone Deacetylase (NuRD) complex and the MEC complex, and functions in larval development, vulval morphogenesis, lifespan regulation, and cell fate determination. To explore the mechanisms involved in the action of LET-418/Mi2, we performed a genome-wide RNA interference (RNAi) screen for suppressors of early larval arrest associated with let-418 mutations. We identified 29 suppressor genes, of which 24 encode chromatin regulators, mostly orthologs of proteins present in transcriptional activator complexes. The remaining five genes vary broadly in their predicted functions. All suppressor genes could suppress multiple aspects of the let-418 phenotype, including developmental arrest and ectopic expression of germline genes in the soma. Analysis of available transcriptomic data and quantitative PCR revealed that LET-418 and the suppressors of early larval arrest are regulating common target genes. These suppressors might represent direct competitors of LET-418 complexes for chromatin regulation of crucial genes involved in the transition to postembryonic development.Mi2 proteins are evolutionarily conserved, ATP-dependent chromatin remodelers of the CHD family that play key roles in stem cell differentiation and reprogramming. In  Drosophila dMi2, together with Polycomb group proteins and the Hunchback transcription factor, regulates the transcriptional activity of the HOX genes during embryonic patterning . The first evidence for a developmental function of NuRD came from studies in mouse embryos, where a lack of the NuRD component Mbd3 compromised the differentiation potential of ES cells . MoleculCaenorhabditis elegans encodes two Mi2 homologs, LET-418 and CHD-3. LET-418 is required for postembryonic development, repression of the germline expression program in somatic cells, proper patterning of the vulva, and lifespan regulation  (FR843), ayIs6[pBH47.70(hlh-8p::gfp) + pMH86(dpy-20(+))] (PD4666), unc-119(e2498::Tc1); wIs51[scmp::gfp + unc-119p::unc-119(+)] (JR667), bnIs1[pie-1p::gfp::pgl-1 + unc-119p::unc-119(+)] (SS747), bnIs1[pie-1p::gfp::pgl-1 + unc-119p::unc-119(+)];let-418(n3536ts) (FR1195), set-26(tm2467) (FR1496), set-9(n4949) (MT16426), set-26(tm2467);let-418(n3536ts) (FR1499), set-9(n4949);let-418(n3536ts) (FR1500), set-26(tm2467); set-9(n4949);let-418(n3536ts) (FR1506), nurf-1(n4295) (MT13649), and nurf-1(n4295);let-418(n3536ts) (FR1495). All C. elegans strains were maintained at 20° using standard conditions (The following strains were used in this study: wild-type (var. Bristol), nditions .Microscopy analyses were performed using a Zeiss axioplan 2 microscope. For brightfield pictures a DIC filter was used, and for fluorescence images the appropriate fluorescence filter was used. A Zeiss AxioCam color camera driven by AxioVision v4.8.2 software was applied for image acquisition.OP50-containing S Medium. During the test, animals were shaken slowly at 25°. After every 24 hr, 100 μl of each sample were transferred to three OP50-seeded NGM plates; the survival rate was first determined and then the animals were kept at 15° to recover. Recovery rates were checked after 5–14 d.Synchronized L4 stage animals were transferred to 25°. After 24 hr, the F1 generation of synchronized embryos was collected and equal amount of embryos were transferred to 20 ml of bacterium-free and HT115 bacteria containing empty L4440 vector were used as control.A previously described feeding technique was applied for RNAi treatment during the study . For thelet-418 worms on HT115 bacteria containing empty L4440 vector) and was shown to be statistically significant .For the genome-wide RNAi screen, RNAi treatment was performed as described above. Two L4 stage worms were added to each well containing different RNAi clones. The plates were then incubated for 8 d at 25°. After the first 24 hr period, the two P generation animals were removed. The F1 progeny was observed. Positive clones were retested in three independent experiments. The level of suppression was determined by comparison with a negative control (let-418(n3536) L4 stage animals carrying the appropriate transgene were transferred to RNAi plates and kept at 25°. Adults were removed after 24 hr of incubation and 40 F1 progeny were analyzed after 48 hr to determine the number of fluorescent cells. The Mann–Whitney U-test was used to determine the significance level.To determine the number of P granule-containing somatic cells, M cells, or V cells, 10 each of wild-type and Synchronized L4 stage animals were treated with RNAi at 25° as described above. After 24 hr, next generation embryos were collected in M9 solution from the gravid adults by hypochlorite treatment and were kept at 25° for 10 hr. Synchronized L1 worms were then put back onto the relevant RNAi plates for 3 hr at 25°. After incubation, larvae were harvested and RNA was isolated using a QIAGEN RNeasy Mini kit, according to the manufacturer’s protocol, combined with Precellys 24 0.5 mm glass beads to break open the animals.http://bioinfo.ut.ee/primer3-0.4.0/primer3/). At least one primer of each primer pair aligned to an exon–exon junction. Primers designed for ama-1 and act-1 genes were used for normalization. The efficiency of the primer pairs was tested by setting a standard curve using a serial dilution of cDNA, and the specificity of the primers was monitored by analyzing the melting curves of each reaction. Data from triplicate reactions were analyzed using a 2−ΔΔCt method. Biological replicates were used to confirm the results. For statistical analysis, we used the Student’s t-test to compare the relative mRNA level of different genetic variants.cDNA was synthesized from total RNA using the QuantiTech Reverse Transcription Kit (QIAGEN). SensiFast SYBR No-ROX Kit (Bioline) was used for qPCR with a Corbett Rotor-Gene 6000 machine driven by Rotor-Gene 6000 v1.7 software. The primers were designed using Primer3 online software (C. elegans online database (http://www.modencode.org) (http://manticore.niehs.nih.gov/pavis2/). Gene ontology (GO) analyses were performed using the DAVID bioinformatics database  mutants, grown at the restrictive temperature of 25°, fail to develop past the first larval (L1) stage. Examination of the mutant phenotype revealed that the two primordial germ cells (PGCs) Z2/Z3 do not divide in let-418 L1 larvae  and the M (mesoblast) cell lineages.The progeny of temperature-sensitive 1 larvae , whereas1 larvae . In fresscm::gfp . However, after close examination of the M cell morphology in let-418 single and daf-16;let-418 double mutants, we observed that a large proportion of let-418 mutant animals were losing the expression of the M cell marker hlh-8::gfp after 3 d of developmental arrest, whereas daf-16;let-418 double mutants or starved wild-type animals did not . This observation suggested that an absence of daf-16 does not suppress mitotic quiescence, but instead prevents the occurrence of some other defects in the let-418 mutant larvae, as indicated by the loss of expression of the M cell marker .Freshly hatched wild-type larvae possess 10 pairs of seam cells. All of them, except the most anterior ones (H0), are blast cells that will divide during development and give rise to different cell types, such as hypodermal cells, neurons, and glial cells  Figure . We analt divide . Furthert divide  . Absence of daf-16 activity prevents the occurrence of some defects in the let-418 larvae, but not the survival rate. Next, we determined the recovery rate of let-418(ts) mutants by returning them to the permissive temperature after different time points. Interestingly, starved let-418(ts) L1 larvae recovered better than fed let-418(ts) mutants, which almost completely lost their potential to resume development after 3 d at restrictive temperature . Obviously, starvation protects let-418 worms and preserves their developmental potential. In summary, our data demonstrated that let-418 L1 larvae stop postembryonic development in a process that looks superficially similar, but is not identical to, that observed in a starvation-induced L1 diapause.In addition to blast cell quiescence, starved L1 larvae exhibit an extended survival rate in the absence of food and, when returned to food, can recover from this diapause state and resume their development. We measured the survival rate of arrested 1 larvae . However18 worms , indicatperature  and exhiLET-418 during the transition to postembryonic development and uncover its regulatory network, we performed a genome-wide RNAi screen to identify suppressors of L1 arrest. Out of the 16,757 genes of the Ahringer library  and let-418(n3536) .set-26 and set-9, that showed 97% identity at the level of their nucleotide sequence. To test if both genes act as suppressors, we generated set-26;let-418 and set-9;let-418 double mutants as well as set-26;set-9;let-418 triple mutants. We found that only the mutation in set-26, but not in set-9, could suppress the L1 arrest phenotype of let-418 . Consistently, the rate of suppression in the set-26;let-418 double mutants was identical to that of the triple mutants set-26;set-9;let-418, suggesting that only set-26, but not set-9, is a let-418 suppressor (set-9 as a suppressor was likely due to the fact that set-9(RNAi) cross-inhibited set-26 expression. set-26 encodes a H3K9 methyltransferase that is involved in the transgenerational sterility of spr-5 mutants lacking H3K4 demethylase activity  gene  gene . Absencelet-418-associated defects.In summary, we isolated mainly chromatin factors in our suppressor screen, suggesting that there is a need to modify the expression of a large number of target genes to suppress let-418 mutants also show ectopic P granule components around somatic nuclei . We tested if our suppressor RNAis were able to reduce the ectopic expression of the reporter construct in a let-418 background. Of the 29 suppressors, 28 significantly decreased the number of cells showing ectopic P granules in let-418 mutants .While P granules appear only in the PGCs Z2/Z3 of wild-type L1 animals, let-418 animals fed on set-26 RNAi grow to the L2/L3 stage, but a large number of P granule-containing cells are still detected  animals, we measured the mRNA level of pgl-1. Consistent with the pie-1p::gfp::pgl-1 reporter analysis, the pgl-1 expression level was elevated in let-418 L1 larvae, but we observed no reduction in let-418;set-26 animals   .hlh-8::gfp, which is expressed only in undifferentiated cells of the M cell lineage (let-418(n3536) animals, the M cell did not divide , although the level of M cell divisions did not fully correspond to the observed developmental stage of the worms. Thus, our data suggest that M cell division and larval growth are uncoupled in let-418 animals. A delay in M cell division compared to the growth rate was previously reported in a daf-2 mutant lacking insulin receptor activity  worms arrest their development at a stage where sex myoblasts are dividing but are not yet differentiating (L3 stage). In addition, these GFP-positive cells are disorganized, indicating defects in sex muscle patterning in let-418;set-26 worms.In ntiating . This obscm::gfp transgene ; However, inactivation of all 29 suppressor genes by RNAi resulted in seam cell divisions to different extents . Similar to our previous observations regarding the M cell divisions, the number of seam cells was lower than expected from the apparent developmental stage of the animal . Altogether, our results show that RNAi of the suppressors can restore blast cell division in a let-418 mutant background to some extent, although none of the worms could reach adulthood.Freshly hatched larvae possess 10 pairs of seam cells. We monitored the developmental fate of the seam cell lineage using animals carrying a let-418 L1 larvae were enriched in germline-specific genes, including those encoding P granule components , and pie-1 (encoding the germline determinant PIE-1) mRNA levels were increased in let-418 larvae   . GO anallet-418 mutants look superficially similar. However, blast cell quiescence in starved larvae, but not in let-418 animals, depends on DAF-16 . This suggests that DAF-16 is involved in both types of developmental arrest, one due to starvation and the other due to an absence of let-418 activity. Therefore, we were interested to know if LET-418 and DAF-16 could share common regulated genes. Indeed, a comparison between the transcription profiles of daf-16 and let-418 mutants revealed a significant number of commonly regulated targets that, following GO analysis, belong to gene categories involved in metabolic pathways  , and tested if they are also coregulated by a subset of our suppressors. We compared the transcriptional activities of these three genes in let-418, nurf-1, htz-1, or set-26 animals with that of let-418;htz-1, let-418;nurf-1, or let-418;set-26 animals. The mRNA levels of dct-3, W08A12.4, and fbxa-165 were increased in let-418 mutants, and this induction was dependent on nurf-1, htz-1, and set-26 activity (LET-418, together with other chromatin-associated factors, is controlling the transcription of a subset of DAF-16 targets that may be involved in larval development.Starved L1 larvae and arrested n DAF-16 . Neverthactivity . This inlet-418 maternal gene activity, let-418 mutants do not initiate postembryonic development. The PGCs and blast cells do not divide. Overall, arrested let-418 worms resemble L1 diapaused larvae, except that they do not survive and recover as well as starved larvae that have been returned to food. By performing a genome-wide RNAi suppressor screen, we could show that let-418 developmental arrest is dependent mainly on genes encoding chromatin factors and on a few other genes exhibiting various functions. All the suppressors identified in this study, except the histone methyltransferase-encoding gene set-26, are able to suppress not only the developmental defects but also the somatic expression of germline-specific genes associated with the lack of let-418 activity. In contrast, set-26 suppresses developmental defects but not the somatic expression of germline genes.In the absence of let-418 worms to initiate postembryonic development was mainly dependent on genes encoding chromatin factors that are members of multi-protein complexes known to activate transcription. Recently, we found that LET-418, together with the histone demethylase SPR-5, blocks the activity of the COMPASS complex to ensure germ cell fate maintenance (LET-418 could control the access of activator complexes, such as COMPASS, but also ISW/NURF, SWR1, KAT8/MOF, and NuA4, to their target genes, thereby maintaining the quiescence and pluripotency of blast cells. Consistent with this hypothesis, we observed that upregulation of some known LET-418-regulated genes, such as the germline genes pie-1, deps-1, and pgl-1, and a subset of DAF-16 targets, is dependent on NURF-1 or HTZ-1 (LET-418 could organize the chromatin structure at target genes, the regulation of which is crucial for developmental transitions.In this paper, we describe that a failure of ntenance , LET-418or HTZ-1  (PassannLET-418 could be responsible for the restriction of gene expression and to ensure responsiveness to developmental cues such as the presence of food.In mammalian cells, Mi2 together with other NuRD components restricts the activity of genes to allow the transition of ES cells from the pluripotent state to lineage commitment . It is pLET-418, is known to repress transcription during development. One possible way to interpret this result is that the suppression effect may occur through MES-4; Polycomb proteins are required for the proper localization of MES-4 exclusively to the autosomes (MES-2/3/6 could lead to a chromosome-wide relocalization of MES-4, resulting in decreased MES-4 activity at LET-418 target genes (mes-2/3/6 is that LET-418 plays a role in the recruitment or localization of Polycomb proteins (LET-418 interacts with MES-2 and can recruit it to the chromatin (let-418 activity could lead to mistargeting of MES-2 and the repression of genes that are not normally bound by the Polycomb complex, thereby resulting in a developmental arrest of let-418 animals. Depletion of PRC2 components may remove this repression and allow development in a let-418 background. This model is also supported by the finding that, in ES cells, Polycomb repressive complex 2 is recruited by NuRD to specific target genes to direct their repression (Among our suppressors, we also found genes encoding members of the Polycomb complex, which, like proteins . This isset-26 was the only suppressor gene that did not suppress the somatic expression of germline genes in let-418 larvae. set-26 is expressed in most, if not all, somatic cells (set-26 gene product methylates lysine 9 of histone H3 in vitro (let-418;set-26 animals. However, such a putative perturbation of the H3K9 methylation pattern would not repress the germline transcription program in somatic cells.in vitro . One posMATH-33 might alter the metabolism of protein turnover to an extent that is sufficient to partially suppress the developmental defects of let-418 (lin-35 (Only five suppressors were identified that did not belong to chromatin factors. As part of the deubiquitylation machinery,  let-418 . Genes i (lin-35 . This suLET-418, are recruited to key developmental genes will contribute to our understanding of the functional importance of chromatin in the response to developmental cues.Altogether, our suppressor screen uncovered a network of chromatin factors that regulate the transition from embryonic to postembryonic development. This transition involves crucial decisions, such as exit from the cell cycle of the blast cells, cell proliferation, and differentiation. Strict control of these processes is critical during development, but also to prevent aberrant proliferation and differentiation during tumorigenesis. Analyzing how these various chromatin factors, together with www.g3journal.org/lookup/suppl/doi:10.1534/g3.116.037747/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Objectives: To compare the safety and intraoperative difficulties of two capsulorhexis techniques for white intumescent cataract: Femtolaser-assisted capsulorhexis and manual capsulorhexis performed in 2-3 stages, with the Utrata forceps.Materials and methods: A prospective comparative study that included 28 eyes divided into 2 equal groups in which capsulorhexis was performed by using the 2 methods. In the first group, the capsulorhexis was executed by using LenSx Femtolaser. In the second group, an Utrata forceps was used to perform a manual 2-3 steps capsulorhexis as follows: a small 2-3 mm capsulorhexis was performed after the staining of the anterior capsule with Trypan Blue along with a good pressurization with viscoelastic substance. The liquefied cortex was aspirated, followed by the enlargement of the capsulorhexis. In some cases, the enlargement was made after IOL implantation. Results: In the Femtolaser group, the capsule was completely detached in 13 cases and only in one case, the capsule had a few bridges which detached easily, without endangering the capsulorhexis integrity. Its size was 4,9 mm in all cases. In the group in which capsulorhexis was performed with the Utrata forceps in 2-3 stages, this was complete, circular and relatively well centered in all cases, but the size varied between 4,5 and 5,5 mm.Conclusions: Femtosecond laser-assisted capsulorhexis was round, well centered and of a desired size of 4,9 mm. The manual capsulorhexis with the Utrata forceps depends on the surgeon’s skill and experience and requires a good local anesthesia, the coloring of the anterior capsule with Tripan Blue, using a large quantity of cohesive viscoelastic substances and sometimes using micro incision forceps for helpful maneuvers. The size and centering of the capsulorhexis are not always identical with the intended ones. Cataract surgery is one of the safest and most efficient surgical interventions, but it is extremely dependent on the surgeon’s experience, especially in challenging cases as intumescent cataract, subluxated lens cataract, hypermature cataract, cataract in the myopic or vitrectomized eye, etc.1].Capsulorhexis is considered the most important step in cataract surgery and offers the lens bag more resistance during hydrodissection, phacoemulsification, and cortex aspiration, reducing the risk of capsular tears and improving the postoperative stability of the IOL. Capsulorhexis was first described in 1987 by Gimbel and Neuhann, as a circular, centered, curvilinear opening in the anterior capsule. These aspects help in maintaining a good positioning of the IOL in the capsular bag while decreasing the incidence of radial tears at the same time [2].In a study by Gavris et al. in 2003, on 70 eyes with intumescent cataract, capsulorhexis skidding frequency was 11.43%, and in one case, 1.43% respectively, the posterior capsule rupture occurred [3], or by implementing a new technological solution like Femtolaser-capsulotomy. Once Lasers were introduced in cataract surgery, a well-controlled and reproducible capsulorhexis was obtained, by laser-tissue interaction called photodisruption. In 2008, Zoltan Nagy performed the first laser capsulotomy during the first femtosecond laser-assisted cataract surgery [4].These unfavorable results determined us to improve our method, by performing a 2-3 stages capsulorhexis as described by Gimbel et al. .In 1991, Gimbel analyzed 2967 cataract cases, out of which 34 were intumescent white cataracts and performed a 2-stages capsulorhexis in these cases. He found that in 4 out of the 34 cases, the anterior capsule tore [8].The very good results we obtained by performing Femtosecond laser-capsulorhexis, as well as by a 2-3 stages capsulorhexis are owed to the surgeon’s experience and her concern in perfecting the capsulorhexis technique in intumescent white cataract, using the proper microsurgery instruments, and implementing new technological solutions [The achieved results confirmed the safety and efficacy of both techniques in performing the capsulorhexis in intumescent white cataracts.The manual capsulorhexis with the Utrata forceps depends on the surgeon’s skill and experience and requires good local anesthesia, coloring of the anterior capsule with Trypan Blue, using a large quantity of cohesive viscoelastic substances, and sometimes using micro incision forceps for helpful maneuvers. Nevertheless, the size and centering of the capsulorhexis are not always identical with the intended ones.Femtosecond laser-assisted capsulorhexis was round, well centered and of the desired size, 4,9 mm respectively.The perfection of the Femtosecond laser-assisted capsulorhexis, along with the surgeon’s increased comfort, make this type of capsulorhexis a superior option or the technique of choice in intumescent white cataract cases, in which the risk of capsulorhexis skidding is greater than in other types of cataract.


In this study, Foster et al. demonstrate that activation of the MRTF–SRF signaling pathway occurs in cancer-associated fibroblasts (CAFs) and is required for their proinvasive and contractile activity. The investigators also identify shared and specific direct genomic targets for MRTF–SRF and YAP–TEAD and show that MRTF and YAP are independently regulated by cytoskeletal dynamics and that this is the basis for their mutual dependence. Both the MRTF–SRF and the YAP–TEAD transcriptional regulatory networks respond to extracellular signals and mechanical stimuli. We show that the MRTF–SRF pathway is activated in cancer-associated fibroblasts (CAFs). The MRTFs are required in addition to the YAP pathway for CAF contractile and proinvasive properties. We compared MRTF–SRF and YAP–TEAD target gene sets and identified genes directly regulated by one pathway, the other, or both. Nevertheless, the two pathways exhibit mutual dependence. In CAFs, expression of direct MRTF–SRF genomic targets is also dependent on YAP–TEAD activity, and, conversely, YAP–TEAD target gene expression is also dependent on MRTF–SRF signaling. In normal fibroblasts, expression of activated MRTF derivatives activates YAP, while activated YAP derivatives activate MRTF. Cross-talk between the pathways requires recruitment of MRTF and YAP to DNA via their respective DNA-binding partners (SRF and TEAD) and is therefore indirect, arising as a consequence of activation of their target genes. In both CAFs and normal fibroblasts, we found that YAP–TEAD activity is sensitive to MRTF–SRF-induced contractility, while MRTF–SRF signaling responds to YAP–TEAD-dependent TGFβ signaling. Thus, the MRF–SRF and YAP–TEAD pathways interact indirectly through their ability to control cytoskeletal dynamics. The interface between mechanical force and gene expression is central to our understanding of normal and transformed cell behavior . Variati. YAP and TAZ, which bind DNA in association with members of the TEAD family of DNA-binding cofactors, were first characterized as effectors of the Hippo growth control pathway , increased with severity of disease stage . The MRTF–SRF target gene data set also exhibited significant overlap with genes overexpressed in myofibroblastic CAFs associated with human pancreatic ductal adenocarcinoma (PDAC)  .Supplemental Fig. S1A). CAFs exhibited elevated expression of the major myofibroblast and contractility markers αSMA/Acta2 and MLC2/Myl9 . Both of these genes are MRTF targets, and their expression in CAFs was MRTF-dependent . In contrast, the TCF (ternary complex factor)–SRF target gene Egr1 was expressed at comparable levels in NFs and CAFs . Consistent with these observations, ChIP analysis demonstrated increased MRTF-A, SRF, and RNA polymerase II (Pol II) recruitment at Acta2 but not Egr1 .In fibroblasts, extracellular signaling through the Rho-actin pathway promotes MRTF nuclear accumulation . AlthougActa2 and the transfected MRTF–SRF reporter was blocked when Rho was inactivated by C3 transferase expression; upon depolymerization of F-actin by latrunculin B (LatB); by the LIMK inhibitor LIMKi3; and by the formin inhibitor SMIFH2 . These inhibitor treatments also reduced MRTF and SRF recruitment to Acta2 as assessed by ChIP .Previous studies have shown that MRTF activation in serum-stimulated fibroblasts is RhoA-dependent and reflects depletion of the G-actin pool as a result of formin or LIM kinase activation . Consistr SMIFH2 D. Acta2 Supplemental Fig. S1F). Consistent with this, treatment of CAFs with Y27632 or blebbistatin, which impair actomyosin contractility, inhibited both Acta2 expression and MRTF ChIP at Acta2 . Treatment of CAFs with the TGFβ family receptor kinase inhibitor SB-431542 abolished Smad2 phosphorylation and induced relocalization of MRTF-A to the cytoplasm . SB-431542 treatment also reduced expression of the MRTF–SRF reporter gene and the MRTF–SRF target genes Acta2 and Myl9 . Conversely, in NFs, TGFβ treatment induced MRTF-A nuclear accumulation  and activated Acta2 and Myl9 in an MRTF-dependent fashion . Taken together, these results establish that MRTF activation is a marker of the CAF activated state and show that TGFβ signaling and contractility are required to maintain it.The establishment and maintenance of the activated state of CAFs and myofibroblasts are promoted by substrate stiffness . Both the MRTF–SRF and the YAP–TEAD target gene sets are enriched for cytoskeletal and ECM remodeling genes and many genes involved in proliferation, signaling, and transcription . Many were also overexpressed in CAFs from the mouse PyMT mammary and KRas/p53 pancreatic cancer models . Comparison of the two data sets thus allowed us to identify potential candidate direct target genes specific for MRTF–SRF alone, specific for YAP–TEAD alone, or shared by both regulators  . All MRTF-only direct genomic targets analyzed were expressed at high levels in CAFs compared with NFs , as was induction of the MRTF–SRF direct target genes Acta2 and Tagln . Conversely, the modest response to serum stimulation of the YAP–TEAD reporter and the YAP–TEAD-specific direct target genes Amotl2 and Ddah1 was inhibited by depletion of both MRTFs and YAP .The mutual dependence of MRTF–SRF and YAP–TEAD target gene expression even at those targets that are directly bound by only one of the transcription factor pairs strongly suggests that their mutual dependence reflects the influence of each on the other's cognate signaling pathway. To examine their mutual dependence more directly, we studied the regulation of MRTF–SRF and YAP–TEAD reporter genes. The elevated activity of an MRTF–SRF reporter in CAFs was sensitive to depletion of not only the MRTFs or SRFs and the Rho-actin inhibitor CCG-203971 A but alsSupplemental Fig. S4). Taken together, these results suggest that the mutual dependence of MRTF and YAP activity arises indirectly and is likely to reflect their influence on each other's signal pathway.We used ChIP to investigate whether the reporter results reflected changes in recruitment of MRTF and YAP to target genes. The enhanced recruitment of MRTF to MRTF–SRF target genes was enhanced in CAFs compared with NFs E. It wasRecent studies have demonstrated physical interaction between MRTF and YAP independent of their DNA-binding cofactor proteins, SRF and TEAD . The ChISupplemental Fig. S5). In CAF1 cells, activity of MRTF-only targets was similar to that seen in CD-treated NFs and was further potentiated by CD . The basal activity of shared MRTF–SRF/YAP–TEAD target genes in CAFs was higher than in CD-stimulated NFs, and CD stimulation had a modest or no further effect . In contrast, although YAP-only target genes were expressed at elevated levels in CAFs, their transcription was suppressed by CD treatment .In CAFs, LatB treatment resulted in the redistribution of both factors to the cytoplasm; in contrast, while CD treatment induced cytoplasmic localization of YAP, it potentiated nuclear accumulation of MRTFs A,B. ConsTaken together, these results suggest that MRTF recruitment and YAP recruitment to DNA are independently regulated. Although our inability to detect mouse YAP by ChIP precluded a direct test of this model using CAFs, ChIP analysis in the human MDA-MB231 cell system corroborated the transcription data. At all target types, MRTF and YAP ChIP signals were abolished by LatB D,E. Howe123-1A is nuclear and active because it cannot bind G-actin . Activation of both MRTF–SRF and YAP–TEAD reporters by MRTF123-1A was dependent on SRF . Together, these data strongly suggest that MRTF123-1A activates the YAP–TEAD pathway by inducing MRTF–SRF target gene expression.In normal fibroblasts, expression of MRTFt on SRF C, and bot on SRF D. In conSupplemental Fig. S7A), its ability to activate the MRTF–SRF reporter was dependent on TEAD . In contrast, activation of shared target genes by MRTF123-1A and 5SA-YAP was partially dependent on YAP and MRTF, respectively . These data are consistent with our assignment of the various target genes to MRTF-only, YAP-only, and shared classes. Moreover, they suggest that constitutively active MRTF activates YAP upstream signal pathways as a result of its ability to activate MRTF–SRF target gene expression and vice versa.Similarly, although the constitutively active YAP derivative 5SA-YAP activated both the YAP–TEAD reporter and YAP–TEAD target genes independently of MRTF . Expression of MRTF123-1A in NFs induced YAP nuclear accumulation, which was inhibited upon depolymerization of F-actin by LatB .We next investigated the mechanistic basis for the mutual dependence of MRTF–SRF and YAP–TEAD signaling. Both pathways are responsive to the state of actin dynamics, so we first investigated whether MRTF by LatB B. Consis123-1A in NF1 cells decreased YAP S127 phosphorylation, which was accompanied by increased Src S416/418 and MLC2 phosphorylation . In NF1 cells, inhibition of myosin (blebbistatin), Src family kinases (dasatinib), or integrinαv (cilengitide) blocked MRTF123-1A-induced YAP S127 dephosphorylation —and contractility itself . Expression of integrins Itgb3 and Itgav, specific targets for cilengitide action, was elevated in CAFs and could be potentiated in NFs by MRTF123-1A expression .These results show that YAP activation by MRTFpression . We showy itself E, and, i123-1A and CD . Consistent with the reported dependence of F-actin assembly on Cdc42EP3 levels . ChIP analysis showed that 5SA-YAP, but not 5SA/S94A-YAP, expression induced LatB-sensitive MRTF-A recruitment to MRTF target genes . 5SA-YAP expression also induced phosphorylation of MRTF-A , which reflects decreased G-actin binding .Consistent with the ability of constitutively active 5SA-YAP to induce F-actin assembly, it also induced nuclear accumulation of MRTF; this was inhibited by LatB and was not induced by 5SA/S94A-YAP, indicating that it involves F-actin assembly and requires activation of YAP–TEAD target genes . Consistent with these data, the ability of 5SA-YAP to potentiate MRTF–SRF reporter activity and Acta2 transcription was sensitive to the LIMK inhibitor LIMKi3, the ROCK inhibitor Y27632, and the TGFβ family receptor kinase inhibitor SB-431542, while its ability to activate the YAP–TEAD target gene Amotl2 was not . Strikingly, elevated TGFβ signaling in CAFs, as assessed by SMAD2 S465/S467 phosphorylation, was strongly down-regulated upon siRNA knockdown of YAP , and, in NFs, its expression was potentiated by 5SA-YAP expression , Smad2/3 , SMA , MLC2 , SRF (sc-335), phospho(T18/S19)-MLC2 , MRTF-A (sc-21558), MRTF-B (sc-47282), pan–ERK , YAP , phospho-Ser127-YAP , TEAD1/TEF-1 , TEAD4/TEF3 (sc-101184), Actin , pan 14-3-3 , histone H3 ; RNA Pol II CTD S5P , Integrin β3 (ab119993), Integrin αV (ab179475), phospho-(Tyr416) Src , Src (2109S), HA high affinity (3F10) , HA-peroxidase high affinity (3F10) , Flag-M2-peroxidase , and Flag . Immunoblotting and immunofluorescence were by standard techniques. F-actin was visualized with Texas Red-X phalloidin (Invitrogen), and nuclei were visualized with DAPI.Supplemental Table S8A. Total RNA was prepared using GeneElute (Sigma) in column format. Following DNase I treatment (Ambion), cDNA synthesis used the Transcriptor first strand cDNA synthesis kit (Roche). qPCR was performed using the ABI 7900 thermocycler (Applied Biosystems) and QuantStudio QS3/QS5 with detection by SYBR Green incorporation (Invitrogen). Relative abundance of template cDNA from intronic sequences was used as a proxy for relative transcription rates and calculated using the ΔCt method, normalizing to the abundance of GAPDH cDNA. For primers, see Supplemental Table S8B.ChIP was as described previously . Three iOrganotypic invasion assays and gel contraction assays were as described . For inhImmortalized NFs and CAFs from the PyMT model  were maihttps://genome.ucsc.edu/cgi-bin/hgLiftOver). The final analysis revealed 725 TSSs that show physical linkage to remote YAP–TEAD sites that, together with 65 TSSs located within 2 kb of a YAP–TEAD site, give a total of 776 potential YAP–TEAD target TSSs.Published YAP–TEAD ChIP-seq data from IMR90 lung fibroblasts  were intOntology analysis was performed as described previously . HypergeP-values were obtained from t-tests with unpaired samples, with a significance threshold of P < 0.05.Analyses were performed using GraphPad Prism software. Mean values and standard error of the mean were generated from the indicated repeats of biological experiments.


Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs), a plant-specific WRKY-type TF, CjWRKY1, and a basic helix-loop-helix TF, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes  were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC) reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay and by a chromatin immunoprecipitation assay. In addition, CjbHLH1 also activated transcription from truncated 4′OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed.The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using  Higher plants produce a large variety of secondary metabolites, which are commonly classified as phenylpropanoids, aromatic polyketides, terpenoids, and alkaloids. These structurally diverse chemicals help protect the plant against pathogens or herbivore attacks and attract pollinators and are also utilized by humans as dyes, flavorings, and pharmaceuticals . In partNicotiana tabacum (Solananceae), monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine in Catharanthus roseus (Apocynaceae), isoquinoline alkaloids (IQAs), berberine in Coptis japonica (Ranunculaceae), sanguinarine in Eschscholzia californica (Papaveraceae) and morphine in Papaver somniferum (Papaveraceae). Among these alkaloids, the biosynthesis of berberine, which is relatively broadly distributed in the plant kingdom and often produced in cultured cells, has been the most intensively investigated (C. japonica cells have been identified and thoroughly characterized (S)-norcoclaurine by (S)-norcoclaurine synthase -norcoclaurine to (S)-reticuline by the sequential reactions of (S)-norcoclaurine 6-O-methyltransferase -coclaurine-N-methyltransferase -N-methylcoclaurine 3′-hydroxylase -3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase -reticuline to berberine by berberine bridge enzyme -scoulerine-9-O-methyltransferase -canadine synthase -tetrahydroprotoberberine oxidase .Among alkaloid biosynthesis, the biosynthetic pathways of several alkaloids have been well investigated at the molecular level due to their chemical uniqueness as well as their economic importance . Examplestigated . Thus, gcterized . These iase NCS; , the conse 6OMT; , (S)-cocse CNMT; , (S)-N-mCYP80B2; , and (S)e 4′OMT; , then -scoase SMT; , (S)-canC. japonica cells, specific transcription factors (TFs) involved in berberine biosynthesis have been isolated and characterized because TFs are key for the regulation of gene expression. Specifically, two general transcriptional activators, CjWRKY1 and the non-MYC2-type CjbHLH1, were shown to regulate the expression of almost all of the berberine biosynthetic enzyme genes present in C. japonica cells  and by the expression of native PsWRKY in P. somniferum  and NtERF189, which belong to the Group IX AP2/ERF subfamily, were isolated and characterized as transcriptional activators in MIA and nicotine biosynthesis, respectively  signaling cascade together with the CORONATINE INSENSITIVE 1 (COI1) and Jasmonate-ZIM domain proteins (JAZs)  . In addis (JAZs) .cis-acting elements in the promoter region of target genes, we aimed to characterize the promoter sequences of biosynthetic enzyme genes. Previous work indicates that WRKY proteins can specifically recognize the W-box DNA sequence motif (TTGACC/T), while bHLH and AP2/ERF TFs mainly recognize the E-box hexanucleotide consensus sequence (CANNTG) and the GCC-box DNA sequence motif (CGCCGCC), respectively  and found that CjWRKY1 activated the CYP80B2 promoter and CjbHLH1 activated the 4′OMT and CYP719A1 promoters in a transient luciferase (LUC) reporter assay  were grown in Linsmaier-Skoog  LUC, 4′OMT promoter::PpLUC, CYP719A1 promoter::PpLUC and 35S::Renilla reniformis (Rr) LUC plasmid vectors were constructed as previously described  was first inserted into the PpLUC vector. Next, the 163 bp CYP80B2 promoter fragments were inserted into the upstream region of the TATA-box using the HindIII/BamHI and XhoI/BamHI restriction enzymes. Mutant plasmids were then generated by inverse PCR-based site-directed mutagenesis using a KOD-plus DNA polymerase .Cauliflower mosaic virus (CaMV) 35S::escribed . TruncatC. japonica protoplasts isolated from 156-S cells cultured for 2–3 weeks. Transformation was performed with polyethylene glycol (PEG) as previously described  after incubation for 24 h at 24°C in the dark. All experiments were performed with three independent biological replicates.Promoter activities were assayed using a Dual-Luciferase reporter assay system  in escribed  using 4 Escherichia coli BL21(DE3) from full-length cDNAs of CjWRKY1 or its mutants cloned into a pGEX-6P-1 vector  at the BamHI and NotI sites. Protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) for 6 h at 28°C. GST-CjWRKY1 proteins were extracted from Escherichia coli cells and purified using Glutathione Sepharose 4B .To determine the direct DNA-binding activity of CjWRKY1 by EMSA, GST-CjWRKY1 recombinant proteins were expressed in 2, 0.5 mM EDTA, 0.05 μg/μl poly(dI-dC)] from a LightShift Chemiluminescent EMSA Kit . After 20 min of incubation at 4°C, the reaction mixtures were separated on a 5% polyacrylamide gel and transblotted onto Hybond-N+ nylon transfer membranes . The shifts in biotinylated probes were detected using a Chemiluminescent Nucleic Acid Detection Module Kit  and using the ImageQuant LAS4010 imaging software .The double-stranded DNA probes for EMSA were prepared from biotin-labeled sense and antisense nucleotides annealed in TEN buffer  for 5 min at 95°C and cooled at room temperature. For EMSA, biotin-labeled probes (20 fmol) and the purified recombinant proteins (3 μg) were mixed with 1x binding buffer  of the CYP80B2 promoter, and the 178 bp sequence between -342 and -164  of the 4′OMT promoter could be important for promoter activities.As shown in CYP80B2 promoter was significantly enhanced by CjWRKY1 expression in a transient LUC reporter assay . CjWRKY1 overexpression also significantly activated the LUC activities of the -418, and -217 truncated promoters of the CYP80B2 gene, while the activities of the -127 and -82 truncated promoters were considerably lowered even with the expression of CjWRKY1, suggesting that the W-box element at -138 might be responsible for the CjWRKY1-mediated induction .Next, we focused on the transcriptional activity of CjWRKY1 to the truncated promoters. As previously described , the LUCCjWRKY1 also enhanced the LUC activities of the -1215, -581, -342, and -164 truncated promoters of the 4′OMT gene , although the enhancement on the -164 promoter was marginal, which highlights the importance of the W-box located at -285. These results suggest that trans-activation of CjWRKY1 on the CYP80B2 and 4′OMT promoters needs at least one critical putative W-box element  while others might be not essential. In contrast, the lack of trans-activation of the CYP719A1 gene by CjWRKY1 suggests that those putative W-boxes found in CYP719A1 might not be functional . Above observation was confirmed by repeated experiments, whereas absolute activity fluctuated at each experiment.The overexpression of CYP80B2 gene were named W1, W2, and W3, and their roles in the transcriptional activity of CjWRKY1 were examined using W-box-mutations (TGACT→TTTTT). As shown in Figure 5A, single, double and triple mutations of putative W-box elements in the full-length CYP80B2 gene generated some variability of relative LUC activities, although these effects were marginal. To study the effects of the putative W-box elements, the -217 sequence was used to evaluate the function of the W2 and W3 elements. Mutations in W2, W3, or both and in the -217 CYP80B2 promoter::LUC vector clearly decreased relative LUC activity with or without CjWRKY1 expression compared to the non-mutated -217 promoter . In particular, the strong reduction of LUC activity in the W2 mutant confirms the important role of the W2 element via CjWRKY1 as previously indicated by the truncated promoter analysis . However, some residual enhancement of LUC activity was still found in the null W-box mutant.Based on the above observation, the three predicted W-box elements found at -227, -138, and -116 in CYP80B2 promoter in more detail, 163 bp of the CYP80B2 promoter between -251 and -88  was added to the TATA-box of the CaMV 35S promoter and the PpLUC gene, and their activities were measured with a dual-LUC reporter assay. Mutations in W2 and W3, and especially in W2, reduced basal promoter activities without CjWRKY1 expression compared to the non-mutated promoter. Interestingly, whereas the effect of CjWRKY1 expression was also reduced with mutations in W2 and W3, considerable LUC activities were still induced by CjWRKY1 . These results confirmed the existence of unexpected LUC activities in the null W-box construct  induced by CjWRKY1 and suggested the possibility that unidentified cis-acting elements are directly or indirectly involved in CjWRKY1-mediated induction.To evaluate the function of the three W-box elements (W1–W3) in the CYP80B2 promoter, a recombinant GST-fused CjWRKY1 protein was incubated with an oligonucleotide probe that contains three tandem copies of the putative W-box (TGACT) found in the W2 nucleotide sequence . Specific binding was detected by EMSA . Further ChIP analysis using overexpressed CjWRKY1-sGFP proteins and anti-GFP antibodies in C. japonica protoplasts  confirmed the amplification of the CYP80B2 gene promoter region by a specific primer designed to detect the W-box element.To confirm the direct binding of CjWRKY1 to the predicted W-box elements in the C. japonica .In addition to CjWRKY1, CjbHLH1 is also a critical regulator of the IQA biosynthesis pathway in japonica . Thus, w4′OMT promoter because CYP80B2 was not responsive to CjbHLH1 and CYP719A1 was not responsive to CjWRKY1 , suggesting that CjWRKY1 and CjbHLH1 function independently through the use of different cis-elements.The interaction of CjWRKY1 and CjbHLH1 was further examined using the C. japonica , 4′OMT (1215 bp upstream sequence) and CYP719A1 (911 bp upstream sequence) managed to activate the LUC reporter gene in C. japonica protoplasts, and two of them showed an induction of LUC activity by either CjWRKY1 or CjbHLH1 co-expression . These results suggest that either isolated promoter regions might not be sufficient or that endogenous expression of TFs may be sufficient for the induction. A clear reduction of transcriptional activity of the 163 bp CYP80B2 promoter region between -251 and -88 with a mutated E-box (CACGTG→ATCGAC) and GCC-box-like (GCAGCC→GCTTCA) elements suggests that the latter interpretation may be more likely . Interestingly, our analysis suggests the involvement of GCC-box and ERF in the regulation of berberine biosynthesis. Although our preliminary transient RNAi assay using available AP2/ERF EST clones . More detailed mutation analysis of W-box elements found in the -217 CYP80B2 promoter sequence further supports the involvement of -138, and -116 W-box elements, although some remaining induction by co-expression with CjWRKY1 was observed even in the null putative W-box reporter construct.The importance of the predicted W-box and E-box elements found in the promoter regions was further confirmed by the LUC activity analysis with truncated promoters and co-expression with CjWRKY1 might interfere/modify the assay system. Furthermore, rice OsWRKY13 is reported to bind not only W-box elements but also the PRE4 element (TGCGCTT), barley SUSIBA2 (HvWRKY46) can bind to a sugar-responsive element (TAAAGATTACTAATAGGAA) as well as the W-box, and NtWRKY12 can bind to the sugar-responsive-like element but not the W-box , the duplicated promoter construct , and the mutation of putative cis-elements . This suggests that further careful investigation of promoter sequences and additional information regarding the preferential interaction of cis-elements with TFs may allow us to further dissect the regulation of alkaloid biosynthesis gene expression. ORCA3, an AP2/ERF transcriptional activator of several MIA biosynthesis enzyme genes. Because the regulation of gene expression is complex and varies among alkaloid species, it is clear that we need additional genome information to study it further. In fact, until now, we did not know how the gene expression of CjWRKY1 and CjbHLH1 was regulated. Recent progress in next generation sequencing technology can help us obtain whole genome sequences of non-model plants such as C. japonica and thus help us elucidate the mechanisms of signal transduction as a result of interactions between TFs and gene promoters in IQA biosynthesis.Our study also indicates that promoter activity can be modified by a combination of YY, TY, and SY prepared materials and carried out the preliminary experiments. YY performed the experiments. YY and FS designed the research and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The data identify and map a “Spacer Promoter” and associated stalled polymerase in the intergenic spacer of the human ribosomal RNA genes, and reveal a very similar enhancer structure to that found in rodents and lower vertebrates.The combination of Chromatin Immunoprecipitation and Massively Parallel Sequencing, or ChIP-Seq, has greatly advanced our genome-wide understanding of chromatin and enhancer structures. However, its resolution at any given genetic locus is limited by several factors. In applying ChIP-Seq to the study of the ribosomal RNA genes, we found that a major limitation to resolution was imposed by the underlying variability in sequence coverage that very often dominates the protein–DNA interaction profiles. Here, we describe a simple numerical deconvolution approach that, in large part, corrects for this variability, and significantly improves both the resolution and quantitation of protein–DNA interaction maps deduced from ChIP-Seq data. This approach has allowed us to determine the  Data from Chromatin Immunoprecipitation (ChIP) combined with Massively Parallel DNA Sequencing (ChIP-Seq) can potentially provide high-resolution maps of transcription and chromatin factor interactions throughout the genome. The absolute resolution of these maps is determined by the size-range of chromatin fragments that are selected during the ChIP step. However, in practice, several other factors limit the resolution achieved by the technique. These include the relative accessibility of the targeted protein–DNA complex  promoters, preinitiation complexes and enhancers that form on the ribosomal RNA genes (rDNA) of mouse and human. Duplications of RPI promoters are found within the rDNA Intergenic Spacers (IGS) of insects, amphibia, and rodents, and are often referred to as “Spacer Promoters”. They were first identified in the rDNA IGS of s laevis  and of Dn plants , and are2, 0.5% NP-40), transferred to Sonication Buffer  and sonicated  for 30 cycles of 30 sec on/30 sec off at high intensity. Each immunoprecipitation (IP) was carried out on the equivalent of 50 × 106 cells in IP Buffer  overnight at 4°. The antibody slurry was prepared with 50 µl A-, 50 µl G-Dynabeads, and 60 µg ml−1 antibody per IP. Immunoprecipitated chromatin was treated with RNaseA and the DNA isolated using 2% Na SDS and 2 mg ml−1 Proteinase-K. Two or more biological replicates were analyzed for each antibody.Cells were fixed with 1% formaldehyde for 8 min at room temperature. Nuclei were isolated using Lysis Buffer  was added as an extra chromosome. For convenience, the origin of the rDNA repeat was displaced to the EcoRI site at 30,493 such that the pre-rRNA initiation site now fell at nucleotide 14,815.The raw fastq.gz files from ChIP and input DNA were checked for quality using FastQC version 0.11.4 . The data were then trimmed using Trimmomatic version 0.33  with optin silico genome for regions most likely to interfere with alignment of rDNA sequences. The “canonical” rDNA repeat sequence (GenBank accession number U13369.1) was fragmented to generate 50 bp nonoverlapping pseudoreads, and these aligned on GRCh38 using Bowtie2 with the −k 10 option. This identified three major regions that would interfere with ChIP-Seq data alignment. The reference genome was, therefore, modified to remove these occurrences; the chromosomes chr22_KI270733v1_random and chrUn_GL000220v1 were removed, and the rDNA sequence present on chromosome 21 was replaced with N . A single copy of the human rDNA repeat (GenBank accession number U13369.1) was then added as an extra chromosome. For convenience, the origin of the rDNA sequence was moved to the EcoRI site at 30,487, such that the pre-rRNA initiation site now fell at nucleotide 12,514.Alignment to the human rDNA proved a little more difficult using the same strategy due to the multiple rDNA sequences already present in version GRCh38. We therefore first searched the human view command of SAMtools . Data deconvolution was achieved by dividing the calculated sample DNA coverage by the appropriate input DNA coverage in order to remove the sequence coverage biases introduced by the sequencing protocol, as described in the main text. At positions where coverage in either data set was of low statistical significance, the deconvoluted data were set to 0 and ignored in subsequent interpretations. The resulting deconvoluted ChIP-Seq data were converted to BedGraph format and visualized using IGV . The manual for the deconvolution protocol and a corresponding Python script can be found at https://github.com/mariFelix/deconvoNorm. Gaussian curve fitting to rDNA promoter subregions was perform using MagicPlot Pro (Magicplot Systems) on data extracted from the BedGraph files.The rDNA chromosome was first extracted from the aligned file with the n 2.25.0 . Each rek 1 option. A Bedgraph of coverage for the 5′ position of each aligned read was then extracted using the genomecov command of BEDTools with the parameters –5, and –strand + (for forward reads) or –strand − (for reverse reads), and visualized using IGV.The 5′ ends of reads from the ChIP-nexus datasets were mapped by first aligning sequences using Bowtie2 as above, but using the unique mapping −https://github.com/mariFelix/deconvoNorm.Mouse strains are available from Jackson Laboratories , and a very limited supply of derived cell lines may also be available upon request. Human cell lines are available from ATCC. The mouse mapping data can be found on ArrayExpress under the accession number E-MTAB-5839. The human data for UBF and RPI in K562 cells can be found on ArrayExpress under the accession number E-MTAB-6032. The HEK293T  and K562 (UBF and input) data from in vivo functions of the RPI transcription factors, as part of an extensive study  in MEFs strongly suppressed sequence enrichment when using antibodies against either factor, RPI engagement being dependent on UBF . Strikinsample coverage/input coverage) for each base position gave an unacceptable level of noise in the mapping profile. To counter this without significantly affecting mapping resolution, we incorporated two steps prior to deconvolution   . ImproveThe overall improvement in factor mapping using the deconvolution protocol can be qualitatively judged by comparing UBF binding across the enhancer repeats as computed using Bowtie2 or the same alignment followed by the deconvolution protocol . For exaTo determine the degree of reproducibility of factor binding deduced using the deconvolution protocol, we compared the binding profiles obtained from different combinations of ChIP-Seq and input DNA biological replicates. R2 between these profiles of 0.47 indicated significant correlation . This strongly suggested that the peaks and troughs of the UBF interaction profile resulted, at least in part, from a preferential positioning of this factor. We counted ∼74 peaks of UBF enrichment within the 47S transcribed region , and these peaks displayed a mean spacing of 170 ± 58 bp. This was roughly consistent with the measured DNA contact length of a UBF dimer  . In a ceexpected . The pose.g., Given its potential importance, it is surprising that a Spacer Promoter has not yet been identified in the human rDNA repeat, though references to its possible existence have been made in the literature e.g., . UBF bine.g., Deconvolution analysis of public and in-house ChIP-Seq data for RPI, TBP, and UBF from human K562 cells further supported this Spacer Promoter mapping. Two peaks of TBP binding were observed on the rDNA, one at the 47S promoter and the other over the prospective Spacer Promoter site, and each TBP peak corresponded to a peak in the UBF binding profile . At highWe previously found that, in mouse, RPI transcription initiated at the Spacer Promoter is arrested ∼40 bp downstream, adjacent to the binding site for the RPI Transcription Termination Factor TTF1 . StrikinDiscussion. The ChIP-exonuclease data further reinforce the notion that, despite the poor primary sequence conservation, the 47S and Spacer Promoters have very similar binary structures.We took advantage of available ChIP-exonuclease mapping data for TBP in K562 . The datXenopus and mouse these repeats possess enhancer or selector-like activities (The DNA lying immediately upstream of the major rRNA promoter in a wide range of eukaryotes has been found to include a variable number of short (∼60–200 bp) sequence repeats . In Xenotivities . Alignmetivities . AnalysiThe potential for very significant improvements in ChIP-Seq mapping resolution afforded by our simple deconvolution protocol were recently demonstrated when the protocol was applied to map transcription factors and chromatin status across the mouse rDNA  that has been suggested to control While the RPI promoters of different organisms from yeast to human show little or no DNA sequence conservation, they do conserve a common functional organization of precisely spaced UPE and core elements, suggesting a similar mode of recognition by the transcription machinery. In fact, we found that realignment of the ChIP-exonuclease (ChIP-nexus) data for TBP . Howeverwww.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300225/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.


Controlling the transcriptional program is essential to maintain the identity and the biological functions of a cell. The Mediator and Cohesin complexes have been established as central cofactors controlling the transcriptional program in normal cells. However, the distribution, recruitment and importance of these complexes in cancer cells have not been fully investigated. Here we show that FOXA and master transcription factors are part of the core transcriptional regulatory circuitry of cancer cells and are essential to recruit M ediator and Cohesin. Indeed, Mediator and Cohesin occupied the enhancer and promoter regions of actively transcribed genes and maintained the proliferation and colony forming potential. Through integration of publically available ChIP-Seq datasets, we predicted the core transcriptional regulatory circuitry of each cancer cell. Unexpectedly, for all cells investigated, the pioneer transcription factors FOXA1 and/or FOXA2 were identified in addition to cell-specific master transcription factors. Loss of both types of transcription factors phenocopied the loss of Mediator and Cohesin. Lastly, the master and pioneer transcription factors were essential to recruit Mediator and Cohesin to regulatory regions of actively transcribed genes. Our study proposes that maintenance of the cancer cell state is dependent on recruitment of Mediator and Cohesin through FOXA and master transcription factors. Transcription factors enforce the cell-specific transcriptional program by binding regulatory elements dispersed throughout the genome236891213Transcription factors exert their control on gene expression through recruitment of a wide variety of cofactors, chromatin modifiers and regulators of the chromosome structure1816The gene expression program controls normal and aberrant biological functions of a cell. Indeed, to reach a disease state, cells often modify their transcriptional program to acquire new identities and functions222528Here we show that FOXA pioneer transcription factors are, with master transcription factors, the core transcriptional regulatory circuitry of cancer cells. These transcription factors are essential to recruit the Mediator and Cohesin complexes to regulatory regions of actively transcribed genes. Taken together, our results suggest that Mediator and Cohesin are connected to the core transcriptional regulatory circuitry through FOXA and master transcription factors to maintain the cancer cell state.Maintenance of the transcriptional program depends on transcription factors and cofactors including Mediator and Cohesin. Indeed, Mediator and Cohesin occupy enhancer regions with master transcription factors to control the transcriptional program and identity of embryonic stem cells30see32The mammalian genome is segmented into functional elements orchestrating transcriptional events and gene expressionAs in normal cells, the gene networks controlling the identity and functions of each cancer cell is unique. To determine the nature of the genes occupied by Mediator, Cohesin and NIPBL, we investigated the associated gene networks in MCF7, HEPG2 and A549. KEGG pathways analyses revealed the association of MED1, SMC1A and NIPBL with genes reflecting the normal and disease functions of the tissues . For exaWhether or not cancer cells are dependent on Mediator, Cohesin and NIPBL to maintain their proliferative state is not known. Therefore, we quantified the cellular response of MCF7, HEPG2 and A549 with proliferation and soft-agar colony formation assays in MED1, SMC1A and NIPBL loss-of-function experiments. For each target, two independent shRNAs were validated for efficiency by Western blot and used in functional assays . In all cis through recognition of a DNA sequence but also in trans through protein-protein interactionsin silico enrichment of the binding motifs found in the HOCOMOCO database in regions occupied by MED1, SMC1A and NIPBL. The 20 most frequent DNA motifs found in regions occupied by MED1, SMC1A and NIPBL were cross-referenced with the list of 20 co-occupying transcription factors (Cofactors are recruited to regulatory elements by transcription factors. To determine the transcription factor(s) recruiting Mediator and Cohesin, we collected publicly available ChIP-Seq datasets for transcription factors  and comp factors . In eachESR1, FOSL2, FOXA1 and JUND. In return, FOSL2, FOXA1 and JUND occupied the promoter of ESR1  and hepatocarcinoma (HEPG2) are known1436STAG2 are frequent in various cancers2852MED12 mutations are found in exon 2 emphasizing alteration of a specific function of the Mediator subunit29The possibility of a general transcriptional mechanism centered on Mediator and Cohesin controlling the transcriptional program of normal and cancer cells raises questions regarding many human diseases. In addition to various types of cancers, genetic alterations in Mediator and Cohesin have been observed in developmental syndromes, neurological disorders and metabolic diseases29If transcriptional regulation is the primary cellular function of Mediator and Cohesin, their importance for other diseases is likely underestimated. Indeed, modulating the activity of regulatory regions is suspected to be an important mechanism leading to human diseases as SNPs are common in enhancer regions24Full materials and methods are available in MCF7  and HEPG2  were grown in DMEM . A549  were grown in F12K medium . Culture medium were supplemented with 10% fetal bovine serum , 100 μM MEM nonessential amino acids , 2 mM L-glutamine , 100 U/ml penicillin, 100 μg/ml streptomycin .For ChIP experiments, MCF7 were kept the last 3 days into DMEM w/o phenol-red  supplemented with 5% of Charcoal/Dextran treated FBS , 100 μM MEM nonessential amino acids , 2 mM L-glutamine  and treated with 100 nM of Beta-Estradiol  for two hours.ChIP-Seq were performed in biological duplicates as previously described20TFF1 and GREB1 loci. For the TFF1 locus, primers GAGATGATGCCACCGTACAC & CCCTCACTCACTTTGAAGCA were used for enhancer 1 (E1), ATCCAGTCCAGGAAGGAGGT & GTCAAGAGAGGAGGCTGTGG for enhancer 2 (E2) and CCGAGTCAGGGATGAGAGG & GGCCTCCTTAGGCAAATGTT for the TSS. For the GREB1 locus, primers GAGCTGACCTTGTGGTAGGC & CAGGGGCTGACAACTGAAAT were used for enhancer 1 (E1), GGGATATGGCTTGTCCATTGT & CATGACACCAGGACCGTAAAG for enhancer 2 (E2) and ACCCAGCAAAACACTTCAGG & ATTCAGCAGTAGCCCTTCCA for the TSS. Two negative control regions (ATGTCAGGCCCATGAACGAT & GCATTCATGGAGTCCAGGCTTT and AGGACCTGCAGCAAACAGAA & TGTCTACATGGGCTAGTGTGCT) were used to calculate fold enrichments.For single gene analysis, primers were designed in the predicted enhancers and promoters regions of https://bitbucket.org/mugqic/mugqic_pipelinest). BigWig files were generated using Samtools 1.2In silico motif searches were performed with the HOCOMOCO database using the PWMEnrich package. For overlaps with chromatin states and between ChIP-Seq datasets, the GenomicRanges R package was used.All ChIP-Seq reads  were aliPlasmid catalog numbers and shRNA sequences are provided in For proliferation and soft agar colony formation assays, cells were transduced with two shRNA per target with the exception of FOSL2 for which only one shRNA was efficiently knocking down all isoforms . BrieflyAccession codes: The data sets supporting the results of this article are available in the NCBI’s Gene Expression Omnibus repository and are accessible through GEO Series accession number GSE76893 .


A small molecule called Sm4 can disrupt interactions involving a transcription factor called Sox18, while having little impact on other members of the SoxF family. Related research article Overman J, Fontaine F, Moustaqil M, Mittal D, Sierecki E, Sacilotto N, Zuegg J, Robertson AAB, Holmes K, Salim AA, Mamidyala S, Butler MS, Robinson AS, Lesieur E, Johnston W, Alexandrov K, Black BL, Hogan BM, De Val S, Capon RJ, Carroll JS, Bailey TL, Koopman P, Jauch R, Smyth MJ, Cooper MA, Gambin Y, Francois M. 2017. Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice. eLife6:e21221. doi: 10.7554/eLife.21221Sox18 is a transcription factor that first came to prominence more than a decade ago when it was discovered that mutations in the gene for Sox18 cause HLTS, a rare human condition that involves loss of hair, build up of lymph fluid and vascular defects . SubsequC. cephalornithos, and was identified by the Queensland-led collaboration in a high-throughput screen for potential Sox18 blockers  and control the rate at which genes are transcribed to produce molecules of messenger RNA. However, the targeting of transcription factors for therapeutic applications can be difficult. This is especially true for transcription factors that reside within the cell nucleus, such as Sox18, because the drug has to pass through the plasma membrane around the cell and then through the double-layered envelope around the nucleus. Drugs that target membrane proteins and cytoplasmic proteins, on the other hand, only have to pass through the plasma membrane.Transcription factors also tend to be part of complex interaction networks, and targeting a single protein-protein interaction in the network is likely to have a relatively small impact, which makes it necessary to target a subset of the interactions in the network. Sox18, for example, is part of an interaction network that involves two other members of the SoxF family of transcription factors – Sox7 and Sox17 – as well as a number of other transcription factors .Transcription factors can work alone or with other proteins, and some carry out their roles as dimers or trimers. Overman et al. – who are based at the University of Queensland and other institutes in Australia, China, the UK and the US – discovered that Sox18 works as a dimer in primary cultured endothelial cells: two Sox18 proteins can bind to each other to form a homodimer, or a single Sox18 protein can bind to another transcription factor (such as Sox7 or MEF2C) to form a heterodimer . MoreoveNeither Sox7 or Sox17 form homodimers: however, they both form heterodimers such as Sox7-RBPJ, Sox7-Sox18 or Sox17-OCT4. Since the expression of all members of the SoxF family appears to be restricted to endothelial cells, each family member can regulate some processes in blood and lymphatic vessels on its own, and regulate other process in tandem with other transcription factors, during both physiological and pathological conditions. In fact, some members of the family cooperate in developmental contexts , and actOverman et al. validate their in vitro findings in zebrafish larvae by demonstrating that Sm4 suppresses genes downstream of the gene for Sox18 and interferes with vascular development. They also showed that the anti-cancer and anti-metastatic effects of Sm4 in a mouse model of breast cancer were caused by the suppression of tumor lymphangiogenesis (the process by which tumors promote the formation of new lymphatic vessels) and the suppression of metastasis via the lymphatic system.The work of Overman, Francois and co-workers elucidates the mechanism of Sox18 and provides a means to pharmacologically inhibit its function. However, there is still much that we do not know about the biological functions of the various transcription factors in the SoxF family: more advanced epigenetic and proteomic approaches using in vivo endothelial cells are needed to make progress in this area. Another challenge is the fact that patients with breast cancer often suffer from a secondary lymphedema, which occurs after removal of tumor tissues and their sentinel lymph nodes, so the risks of Sm4 or any other small molecule exacerbating this condition needs to be evaluated.


Fructose-1,6-bisphosphatase (FBP1) plays an essential role in gluconeogenesis. Here we report that the human FBP1 gene is regulated by two liver-enriched transcription factors, CCAAT-enhancer binding protein-α (C/EBPα) and hepatocyte nuclear factor 4α (HNF4α) in human hepatoma HepG2 cells. C/EBPα regulates transcription of FBP1 gene via binding to the two overlapping C/EBPα sites located at nucleotide -228/-208 while HNF4α regulates FBP1 gene through binding to the classical H4-SBM site and direct repeat 3 (DR3) located at nucleotides -566/-554 and -212/-198, respectively. Mutations of these transcription factor binding sites result in marked decrease of C/EBPα- or HNF4α-mediated transcription activation of FBP1 promoter-luciferase reporter expression. Electrophoretic mobility shift assays of -228/-208 C/EBPα or -566/-554 and -212/-198 HNF4α sites with nuclear extract of HepG2 cells overexpressing C/EBPα or HNF4α confirms binding of these two transcription factors to these sites. Finally, we showed that siRNA-mediated suppression of C/EBPα or HNF4α expression in HepG2 cells lowers expression of FBP1 in parallel with down-regulation of expression of other gluconeogenic enzymes. Our results suggest that an overall gluconeogenic program is regulated by these two transcription factors, enabling transcription to occur in a liver-specific manner. The liver plays an important role in maintaining glucose homeostasis , 2. Duricis-acting elements that mediate basal transcription of FBP1 have been reported  supplemented with 28 mmol/l NaHCOeviously . The traeviously .5 HepG2 cells were seeded into a 6-well cell culture plate before transfection with 1.6 pmol of C/EBPα-pcDNA3, hHNF4α-pcDNA3 or empty vector in the presence of 10 μl of Lipofectamine 2000 reagent. The transfected cells were maintained as described above before being harvested for Western blot analysis and EMSA.For overexpression studies, 5x102, 10 mM KCl, 0.5 mM DTT, 1x protease inhibitor (Roche) and incubated on ice for 15 min. Fifty microlitres of 10% (v/v) NP-40 was added to the cell suspension before centrifugation at 3000x g at 4°C for 3 min. The pellet was resuspended in 50 μl of cold buffer 2 containing 20 mM HEPES-KOH buffer, pH 7.9, 25% (v/v) glycerol, 0.2 mM EDTA, 10 mM KCl, 1.5 mM MgCl2 and 420 mM NaCl, and incubated on ice for 40 min. After centrifugation at 12,000 x g at 4°C for 15 min, the supernatant was collected and stored at -80°C until used. The 3’-labeled biotinylated oligonucleotides  were annealed with their unlabeled complementary strands in 1x annealing buffer . The oligonucleotides used in EMSA are shown in 2), 2 μg of poly (dI-dC), 0.05% NP-40, 2.5% (v/v) glycerol, 60–480 fmole of biotinylated labeled double-stranded oligonucleotide probe and 5 μg of nuclear extract or 0.2 μg of purified 6xHis hHNF4α  for the nuclear receptors (NR)   . These ts effect . It wouls effect .HNF4α is a liver-enriched transcription factor, belonging to member of hormone nuclear superfamily and highly expressed in liver as is C/EBPα . In livein vivo or in cultured hepatocytes also affect expression of all gluconeogenic enzymes [HNF4α has previously been reported as the vitamin D3-responsive element (VDRE)/retinoic acid responsive element (RAR) that mediates vitamin-D/retinoic acid–induced FBP1 expression in monocytes , 37. It  enzymes , 64In summary we show that C/EBPα and HNF4α are the two important transcription factors that regulate expression of human FBP1 expression in HepG2 cells. This transcriptional activation is mediated through binding to -227/-218 and -218/-209 C/EBPα binding sites and through -566/-554, and -212/-198 HNF4α binding sites, respectively. Suppression of expression of both transcription factors results in a marked decrease in expression of all gluconeogenic enzymes.