We mapped differentiation- and maturation-associated changes in transcription factor binding sites enriched at each stage and observed overrepresentation of AP-1 gene family motifs in barrier-deficient samples. Transcription of TSLP, an important effector of immunological memory in response to allergen exposure, was dramatically elevated in our barrier-deficient samples. We identified dynamic DNA elements that correlated with TSLP induction and may contain enhancers that regulate TSLP. Two dynamic regions were located near the TSLP promoter and overlapped with allergy-associated SNPs rs17551370 and rs2289877, strongly implicating these loci in the regulation of TSLP expression in allergic disease. Additional dynamic chromatin regions ~250kb upstream of the TSLP promoter were found to be in high linkage disequilibrium with allergic disease SNPs. Taken together, these results define dynamic chromatin accessibility changes during epidermal development and dysfunction.To identify putative gene regulatory regions that respond to epidermal injury, we mapped chromatin dynamics in a stratified human epidermis during barrier maturation and disruption. Engineered skin substitutes (ESS) cultured at the air-liquid interface were used as a model of developing human epidermis with incomplete barrier formation. The epidermal barrier stabilized following engraftment onto immunocompromised mice, and was compromised again upon injury. Modified formaldehyde-assisted isolation of regulatory elements (FAIRE) was used to identify accessible genomic regions characteristic of monolayer keratinocytes, ESS  A major role of the epidermis is to act as a barrier that protects against external pathogens and prevents surface water loss. Disruption of the barrier induces an immune response to destroy invading organisms and create immunological memory against specific antigens. Dysregulation of this process can involve ectopic activation of the Th2 immune system and lead to allergic diseases including atopic dermatitis, asthma, and allergic rhinitis. Downstream of barrier disruption are a series of cellular responses, including changes in gene expression. Analysis of chromatin accessibility dynamics can identify potential regulatory regions that mediate the transcriptional response and provide insight into the mechanisms that link barrier dysfunction with immune sensitization.TSLP), to activate the local immune system [Key components of the epidermal barrier include cross-linked lipids and proteins, such as Filaggrin, Loricrin, and late cornified envelope (LCE) proteins. When the physical barrier is breached, the epidermis also acts as a first-response immune organ. Keratinocytes below the barrier express pattern recognition receptors ; when stimulated by pathogens or allergens they produce of cytokines, including interleukins IL-1, -6, -25, and -33, and thymic stromal lymphopoietin (e system  and recrTSLP in humans: the short form (sfTSLP) is constitutively expressed in skin and may act as an anti-microbial protein [TSLP (lfTSLP) is an IL7–like cytokine that binds to a receptor dimer consisting of the IL7 receptor alpha chain and TSLP receptor [lfTSLP acts directly on dendritic cells, type 2 innate lymphoid cells, and eosinophils, and promotes conversion of naïve T-cells into Th2 CD4 cells to create systemic immunological memory for an antigen [lfTSLP signaling in mice with epidermal barrier defects prevents the progression from skin disease to airway inflammation and hyperreactivity [lfTSLP transcription have been proposed [Loss of epidermal barrier integrity and over-activation of the Th2 arm of the immune system are characteristic features of atopic dermatitis (AD). In human disease, the progression from atopic dermatitis to asthma to allergic rhinitis is often referred to as the “atopic march”  and occu protein . The lonreceptor . In huma antigen . Blockinactivity –16. Whilproposed , it remalfTSLP induction (henceforth referred to as TSLP), we analyzed genome-wide chromatin accessibility during multiple stages of epidermal barrier development and disruption. Changes in chromatin accessibility reflect an accumulated effect of covalent histone modifications [To investigate the molecular pathways operating during differentiation and directly upstream of epidermal injury responses, including ications , nucleosications , chromatications , and traications . This inications , 22–24. ications  to succeTSLP  could serve as a model of human skin during barrier maturation and disruption. ESS consist of stratified primary human epidermal keratinocytes cultured on dermal fibroblasts at the air-liquid interface (ALI) , 27 and TSLP . Barrierg injury . Expressr system . TherefoTSLP expression [TSLP mRNA. To capture the chromatin conformation in ESS without introducing excessive experimental artifacts, we chose to use formaldehyde-assisted isolation of regulatory elements (FAIRE) [Protease activity is known to induce pression –30. Acco (FAIRE) , 31, 32, (FAIRE) ). Using  (FAIRE)  we generTo determine whether FAIRE peaks were associated with tissue-specific gene expression, we obtained lists of genes from the Human Protein Atlas  that eitWe next asked whether the genes associated with FAIRE peaks also reflected tissue-specific enrichment. Homer annotation  was usedin vitro ESS); Intact barrier (found in Graft but not ESS in vitro or Tape-stripped graft); and Barrier-deficient epidermis   . Most FAd graft) . These rThese dynamic peaks represent potential regulatory regions that respond to barrier damage and induce transcriptional responses. To investigate which molecular pathways might be involved in this process, we analyzed transcription factor (TF) binding motifs specifically enriched under dynamic peaks in basal Keratinocytes, ESS, Grafted ESS, or Tape-stripped graft using Homer’s motif enrichment software  and the TSLP regulatory regions we inferred the most likely transcriptionally associated domain (TAD) for the TSLP locus on Ch5q22.1 using published Hi-C (genome-wide chromosome conformation) data obtained from multiple cell types [SLC25A46, WDR36, and sfTSLP) did not significantly change in our samples TSLP regull types , 41. The samples , but theTSLP expression (ESS and Tape-stripped graft) but were absent from stable epidermis with an intact barrier (Graft) or monolayer cell cultures that do not express TSLP  peaks present only in stratified tissues; and Barrier-deficient peaks which were present in the samples with insufficient barrier function and hylation .TSLP TAD  were located within Barrier-deficient FAIRE peaks near the TSLP promoter (2 > 0.8) LD with rs17551370 and one in strong LD with rs2289277  between disease-associated SNPs and SNPs in this FAIRE peaks. To further delineate which peaks may be associated with allergic or atopic disease, we investigated the association between dynamic FAIRE peaks and specific single nucleotide polymorphisms (SNPs). Sixteen allergic phenotype- associated SNPs were identified within the TSLP TAD , Table 1s2289277  Figs. Ins2289277  Figs. Inin vitro monolayer differentiation [in vivo human development and disease.The skin is a complex organ that serves as the interface between an organism and its environment. Skin keratinocytes respond to epidermal disruption by producing a variety of pro-inflammatory cytokines, neurotransmitters, and hormones . Defectsntiation –59, but in vivo. Interestingly, different stages of epidermal development were defined more by their chromatin signature than the genes annotated to each FAIRE peak. These results concur with those of Cavazza, et al. [in vitro keratinocyte differentiation and found that while there was considerable overlap in the promoters transcribed in progenitor and differentiating keratinocytes, the active enhancers were often unique to a particular stage. However, we did observe that when FAIRE peaks present in Keratinocytes and ESS were filtered from Stable graft data, the GO term enrichment for epidermis-related categories diminished, suggesting that chromatin near genes commonly associated with epidermal differentiation is already open in cultured keratinocytes. This interpretation is supported by the presence of histone 3 lysine 4 monomethylation (H3K4me1) in ENCODE NHEK [Our study revealed characteristic patterns of open chromatin during multiple stages of skin barrier development and function, starting with human keratinocytes grown in a monolayer, stratified at the air-liquid interface, and after stable engraftment , et al. , who anaODE NHEK , a histoin vivo to ESS in vitro revealed changes in maturation of melanocyte and patterns associated with neural development, which may be due to rare melanocytes, cells of neural crest origin that were undetectable in ESS in vitro but expanded after grafting to form spots of pigment visible in some grafts in vivo  and ECM remodeling . Performing grafting experiments on immunocompromised mice housed in a barrier facility neutralized the confounding influence of many immune cell-derived cytokines [TSLP induction, it is unclear which are required for the increase in TSLP expression seen in human disease.TSLP is a key component in the inflammatory response to barrier damage. TSLP can induce immunological memory leading to the atopic march, and has proven potential in inducing anti-tumor immunity in mice , 67 and rmatitis . TSLP caroteases , 46, Vitroteases –72, Tollroteases –78, and roteases , 80. Howroteases  as well roteases  are dispytokines , 83, allTSLP TAD; Epidermal and 3D peaks could represent enhancers that are necessary for sfTSLP and/or TSLP transcription, and Barrier-deficient peaks may be responding to the sensor that detects epidermal barrier breach and induces TSLP. Two Barrier-deficient peaks were identified near the TSLP promoter, both of which overlapped allergy associated SNPs ; th70 [TSLP . In bothTSLP locus that likely play a role in regulating TSLP transcription in allergic disease. By looking at the intersection of allergic disease associated SNPs and Barrier-deficient peaks in our ESS model, we markedly reduced the number of regions which could contribute to regulation of TSLP after barrier injury. In conjunction with published gene expression profiles and chromatin dynamics of keratinocytes responding to calcium in 2D culture, these data will enable the assembly of a more complete image of the regulatory events during human epidermal differentiation, barrier maturation, and response to injury.We successfully isolated open chromatin from a stratified, stable human epidermis and identified DNA regions uniquely associated with different stages in epidermal development and injury response. In particular, we identified key regions near the ^6/ cm2 on a collagen matrix. 24 hours later primary human epidermal keratinocytes were added at 1x10^6/ cm2. The collagen and cells were cultured at the air-liquid interface for 14 days in University of Cincinnati Dermatology Medium-1 (UCDM-1) with daily media changes [De-identified skin from surgical discard from reduction mammoplasty was collected from elective surgeries at the University of Cincinnati (UC) Medical Center. Because the samples were de-identified and no Protected Health Information (PHI) was collected, the UC Institutional Review Board (IRB) determined that this activity does not constitute human subjects research and is exempt from requirements for informed consent according to 45CFR46.101(b)(4). Engineered human skin substitutes (ESS) were prepared as previously reported  from 2 s changes .NIH-Lystbg-JFoxn1nuBtkxid). Mice were housed four to a cage in pressurized Individually Ventilated Cages with forced ventilation and water directly to each cage in the University of Cincinnati laboratory animal facility. They were provided with unrestricted access to standard chow and water. Deep bedding and nesting material were included in each cage.ESS were grafted onto 14 6–8 week old female Charles River NIH-III nude mice . Part  of each cage was maintained on a heating pad to warm it; as a safety measure, part of the cage was not on the heating pad. Post-operative analgesics were not provided as these drugs alter or inhibit the wound healing process and would reduce the value of those studies. Additionally, the surgical site is a full thickness excision of skin with sharply-defined edges. Thus, the excised area no longer has functioning nerves and there is no gradient of injury between the excised region and surrounding skin. If the grafted sites are touched, the mice show no signs of distress.Post-operative mice were monitored daily by the investigator as well as an animal facility technician. Dressing materials were closely inspected for evidence of chewing or scratching, and mice were re-dressed if necessary. Any mice demonstrating signs of pain or distress including difficulty in breathing, reduced mobility, hunching, rapid weight loss, not eating, were euthanized and excluded from the study.Bandages were removed 2 weeks post-surgery and the grafts were allowed to heal for an additional 4 weeks. 6 weeks after grafting, the mice were divided into two groups: untreated  and tape-stripped, n = 7. Grafts from each cell donor were divided evenly between the groups. For tape-stripping, mice were anesthetized with isoflurane, using the same toe-pinch standard as above along with ophthalmic ointment. Initial trans-epidermal water loss (TEWL) measurements were recorded with a VapoMeter . Fresh 0.75 in square strips of 3M Scotch Magic tape were applied and rapidly removed in succession, with breaks for TEWL measurements. Tape-stripping continued until TEWL readings either reached a plateau, exceeded 4 times the original TEWL measurement, or the number of tape strips equaled 80. None of the mice demonstrated any distress during or after the tape-stripping process. Tape-stripped mice were returned to sterile cages. Three hours later, mice were euthanized and 4 mm punch biopsies were collected and flash frozen in liquid nitrogen for future RNA isolation. Tissue at the intersection of human ESS graft and mouse skin was fixed in 4% PFA for histology analysis. The remaining graft tissue was removed and flash-frozen for FAIRE analysis. Animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health with the approval of the University of Cincinnati Institutional Animal Care and Use Committee.To isolate RNA from ESS and skin samples, fresh or frozen tissue was dissociated with a pellet pestle in RLT lysis buffer (Qiagen ID: 79216) with 1% beta-mercaptoethanol and passed through a QIAshredder column (Qiagen ID: 79656). RNA was purified using the Qiagen RNeasy Mini kit (Qiagen ID: 74106). cDNA was synthesized using the SuperScript II reverse transcriptase (Invitrogen 18064–022) with 50–200 ng RNA. Quantitative PCR was performed with iTag Universal SYBR Green Supermix (BioRad 172–5121). Primer sequences are listed in Frozen ESS were pulverized to a fine powder in liquid nitrogen and thawed in 1 mL 1% methanol-free formaldehyde . Samples were transferred to a 15 mL tube and an additional 7 mL 1% methanol- free formaldehyde was added. After 30-minute fixation at room temperature on a rotator, Glycine was added to a final concentration of 0.13 M and the sample placed on the room-temperature rotator for 10 minutes. The tubes were centrifuged for 3 minutes at 700 x g at 4°C and the pellet was resuspended and washed three times in cold PBS at 4°C for 5 minutes. After the final wash, the fixed tissue was resuspended in 1 mL of sonication buffer containing 5% SDS and 10 mM Tris-HCl pH 8.0; 1 mM EDTA; 0.5 mM EGTA, 100 mM NaCl; 0.33% TritonX-100; 0.4% Sarkosyl; 0.03% Deoxycholate. Samples were kept on ice and rotated through 4–8 sonication cycles using the Branson 250 Digital Sonifier with a 1/8 inch doublestep tip (Branson Ultronics 101063588 (sonicator), 101148063 (tip). Each cycle consisted of 1 minute of sonication split into 1 second on/ 1 second off intervals  at an amplitude maximum of 50%. Following sonication a 50 μl aliquot was taken to test sonication efficiency. The aliquot was incubated at 55°C with ProteinaseK and RNase A (0.2 mg/ml each) for 1 hour to reverse the crosslinks. The DNA was isolated by phenol-chloroform extraction and ethanol precipitation and analyzed on a 1% agarose gel. Once DNA sonication reached the desired level of fragmentation (approximately 150–600 bp), the sonicated tissue was transferred to a 1.7 mL tube and spun for 10 minutes at 10,000 x g. The resulting supernatant containing cross-linked proteins and DNA was separated from the pellet containing leftover tissue and debris and transferred to a new vial. The FAIRE phenol-chloroform extraction and DNA isolation then followed as described in .http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc) and the reads were aligned to the human genome hg19 with Bowtie 2 using default parameters [FAIRE DNA sequencing libraries were assembled using the Illumina low throughput sequencing adaptors and indexing primers (see TruSeq DNA Sample Prep Guide Illumina 15026486 C). Following quantification with Q-bit , 40 ng DNA were used for the sequencing library. Library preparation was as follows: DNA fragments underwent end repair with T4 DNA Polymerase (New England Biolabs (NEB) M0203L), Klenow DNA Polymerase (NEB M0210L), and T4 Polynucleotide Kinase (NEB M0201L). Following purification with Agencourt AmPure XP DNA-binding beads (Beckman Coulter A63880) in polyethylene glycol (PEG) at a 1.9:1 bead: DNA volume ratio, adenine bases were added to the ends of each fragment with Klenow exonuclease (NEB M0212L). Pre-annealed adaptors were ligated overnight at 16°C with T4 DNA Ligase (NEB M0202L). Unligated adaptors and adaptor dimers were removed with an AMPure XP bead purification (1.2:1 bead: DNA) performed twice. PCR following adaptor ligation was performed with Phusion High-Fidelity DNA Polymerase (ThermoScientific F-530L) using unique Illumina TruSeq DNA LT indexing primers for 12–14 amplification cycles. Following PCR, amplified DNA underwent a final AmPure XP bead purification at a 1.1:1 or 1:1 PEG to DNA volume ratio. A small amount was run on the Fragment Analyzer  to confirm successful removal of adaptors and other small DNA fragments. Sequencing was performed on the Illumina Hi-Seq2500. The quality of the resulting FASTQ files was analyzed with FastQC  were merged to better differentiate consistent FAIRE peaks. Data were analyzed using Homer software  as well FAIRE peaks were identified using Homer findPeaks  with theTo define the 5 subclasses of FAIRE peaks , we first ran intersect from BEDTools  using thMotif analyses were performed using the Homer motif enrichment toolkit . To geneTSLP TAD  catalog  and fromTSLP TAD . The loc2 from 1000 Genomes phase 1 data of CEU (European) founders [2 greater than 0.8. Variants which were in strong LD with disease associated variants and were within a FAIRE peak were considered to have putative evidence of disease relevant regulatory effect [We estimated LD using Rfounders . We defiy effect .S1 FigSLC25A46, WDR36, as well as isoform specific primers for sfTSLP. T-test comparisons of expression levels of each transcript among samples revealed no statistically significant differences (p>0.05). PCR primer sequences are in (TIF)Click here for additional data file.S2 FigAnalysis of FAIRE signal from Keratinocyte, ESS, Graft, and Tape-stripped ESS, as well as ENCODE NHEK, GM12878, HUVEC, and HepG2 cells, within 2.5 kb of the transcription start sites (TSS) of genes either: 1. elevated in a specific tissue, 2. with mixed expression in the designated tissue and others, 3. ubiquitously expressed, 4. not expressed in the specific tissue type, or 5. not detected in any tissue. Graphs for genes from spleen and brain are shown.(TIF)Click here for additional data file.S3 FigTSLP TAD showing SNPs that are associated with allergic disease (red) and SNPs located within in dynamic FAIRE peaks (black). Signal track from ESS is used to show peak location. Bold black SNPs are in LD (R2 > 0.8) with a disease-associated SNPs. Bold red SNPs are associated with disease and present in a dynamic peak. In the LD plot below, black represents an R2 of 1 (100%), and white is no linkage. Squares boxed in red indicate loci of high LD between a SNP in a dynamic peak and an allergy-associated SNP.The (TIF)Click here for additional data file.S4 Fig2 on a scale from 0–100, with black indicating a value of 100 (perfect linkage).LD block as in (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 TableThe GREAT tool  was used(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Appendix(XLSX)Click here for additional data file.S2 Appendix(XLSX)Click here for additional data file.in vitro  reported that many promoter-enhancer contacts were pre-established in undifferentiated keratinocytes. This observation strengthens our conclusion that many differentiation-specific genes are embedded in chromatin already open within basal keratinocytes. In addition, capture Hi-C data recorded the presence of long-range interactions between some of the putative enhancers identified by FAIRE and the TSLP promoter. Enhancer/promoter interactions within stable, grafted human epidermis remain to be determined.As this manuscript was being reviewed, a genome-scale analysis of chromatin architecture during calcium-induced keratinocyte differentiation


Drosophila metamorphosis and identified the evolutionarily-conserved homeodomain protein Cut as a master switch in this process. A steep Wnt/Wingless morphogen gradient intersects with a pulse of steroid hormone ecdysone to induce cut expression in a subset of midgut progenitors and reprogram them into renal progenitors. Molecularly, ecdysone-induced temporal factor Broad physically interacts with cut enhancer-bound Wnt pathway effector TCF/β-catenin and likely bridges the distant enhancer and promoter region of cut through its self-association. Such long-range enhancer-promoter looping could subsequently trigger timely cut transcription. Our results therefore led us to propose an unexpected poising-and-bridging mechanism whereby spatial and temporal cues intersect, likely via chromatin looping, to turn on a master transcription factor and dictate efficient and precise lineage reprogramming.Understanding how cellular identity naturally interconverts with high efficiency and temporospatial precision is crucial for regenerative medicine. Here, we revealed a natural midgut-to-renal lineage conversion event during  As an embryo develops, an organism transforms from a single cell into an organized collection of different cells, tissues and organs. Regulated by genes and messenger molecules, non-specialized cells known as precursor cells, move, divide and adapt to produce the different cells in the adult body.However, sometimes already-specialized adult cells can acquire a new role in a process known as lineage reprogramming. Finding ways to artificially induce and control lineage reprogramming could be useful in regenerative medicine. This would allow cells to be reprogrammed to replace those that are lost or damaged. So far, scientists have been unable to develop a clear view of how lineage reprogramming happens naturally.Here, Xu et al. identified a cell-conversion event in the developing fruit fly. As the fly larva develops into an adult, a group of cells in the midgut reprogramme to become renal cells – the equivalent to human kidney cells. The experiments revealed that a combination of signals from a cell messenger system important for cell specialization  and the hormone that controls molting in insects, activate a gene called cut, which controls the midgut-to-renal lineage reprogramming.Together, Wnt and the hormone ensure that cut is activated only in a small, specific group of midgut precursor cells at a precise time. The reprogrammed cells then move into the excretory organs, the renal tubes, where they give rise to renal cells. Midgut precursor cells in which cut had been experimentally removed, still traveled into the renal tubes. However, they failed to switch their identity and gave rise to midgut cells instead.Further examination revealed that both Wnt and the ecdysone hormone are needed to activate the cut gene. This is probably achieved by creating loops in the DNA to bring together the two distantly located key regulatory elements of cut gene expression. If this mechanism can be seen in other contexts it may be possible to adapt it for medical purposes. The ability to reprogramme groups of cells with high specificity could transform medicine. It would make it easier for our bodies to regenerate and repair. Classical regenerative strategies are facing challenges, including difficulties associated with the acquisition, delivery and integration of proper cell types into a complex milieu of tissues. In vivo lineage reprogramming, conversion of a highly specialized cell into the desired cell identity, has therefore emerged as an alternative and promising regenerative strategy . HoweverDrosophila metamorphosis. Drosophila excretory system, so-called Malpighian tubules, are two pairs of tubules converge through common ureters onto midgut-hindgut junction  and identified the homeodomain protein Cut as a master switch dictating this lineage reprogramming event. Cut is an evolutionarily-conserved homeodomain-containing transcription factor that has been shown to regulate various developmental events in modeling .cut expression in a subset of midgut progenitors and reprogram them into renal progenitors. Mechanistically, the Wg morphogen gradient, through its pathway effector TCF/β-catenin, determines the pool of future renal progenitors, presumably by poising a distal cut enhancer for timely activation. On the other hand, the hormone ecdysone-induced BTB-Zinc finger protein Broad determines the timing of lineage conversion by physically interacting with enhancer-bound TCF/β-catenin complex and likely bridging the distal enhancer and promoter region of cut through its self-association. Such long-range enhancer-promoter looping could subsequently trigger timely cut transcription. Thus, integration of spatial and temporal cues by a master cell identity switch, likely through a chromatin looping mechanism, orchestrates natural lineage reprogramming with temporal and spatial precision.Here, we show that a steep Wnt/Wingless (Wg) morphogen gradient  at the mesg-Gal4, UAS-CD8-GFP, tub-Gal80ts system to drive RNAi expression, transferred animals from permissive temperature (18°C) to restrictive temperature (29°C) at mid third instar larval stage, approximately 48 hr before the lineage reprogramming event occurs, and analyzed the renal phenotypes at early adult stages. Intriguingly, we found that midgut progenitor-specific knockdown of the transcription factor Cut resulted in lack of the entire lower ureter region  that differentiate into Cut- EEs and ECs in the midgut  differentiate into Cut+ principal cells (PCs) on the renal tubules  midguts   system  expression, transferred animals from permissive temperature (18°C) to restrictive temperature (29°C) at late third instar larval stage and analyzed the renal phenotypes at early adult stages . The adult escaper flies lacked the whole lower ureter region  system . We usedt stages . Upon esesg-Gal4 . In wildr region , hintingr region , Esg- laid, Cut- , and expid, Cut- , a typiccut depletion, Esg+ progenitors along renal tubules behaved essentially the same as MPs at pupal stages  thetor Rab3 , synaptitor Rab3 , and sector Rab3  tor Rab3 .+ EEs or Pdm1+ polyploid ECs were present in wild type MARCM clones in the midgut   . While m NRT-Wg) , it fail NRT-Wg) , indicatwg-Gal4, wg-lacZ or Wg signaling reporter Fz3-GFP at the midgut-hindgut boundary remained relatively constant from second instar larval to early pupal stages  (+ progenitor-specific depletion of the Broad complex (Br-C)    . Since E(EcR-DN) , we consx (Br-C) , a crucix (Br-C) , phenocox (Br-C) . These o decline . Togethecut induction. Furthermore, Wg pathway activity, as indicated by the Fz3-GFP reporter, was highly responsive to a reduction in Wg signaling, but remained unaltered upon downregulation of ecdysone signaling  assays. The Br-C gene locus encodes four distinct splicing isoforms, Br-Z1, Br-Z2, Br-Z3 and Br-Z4, which share a common N-terminal core domain but have distinct C-terminal zinc-finger domains   cut intron2-enhancer contains closely-spaced putative TCF- and Br-Z2-binding sites  results demonstrated a direct and sequence-specific binding of TCF and Br-Z2 to their putative binding sites in the cut promoter or intron2-enhancer region  cuting site ; and (3)ing site , which iing site . To testr region . Next, wr region . Furtherr region . Finallyture RPs , indicatpression , demonst+ progenitors migrate normally, yet fail to switch identity and differentiate into midgut cells along renal tubules  or Nipped-B  of osophila . Compellosophila . Therefocut is an unusually large gene harboring a long and complex enhancer region spanning more than 150 kb. Such a long enhancer region can be subdivided into small segments responsive to different stimuli or signals in diverse tissues or organs at distinct developmental stages. Therefore, akin to neurons with extensive and complex arbors, the extra-long and segmented enhancer region of cut receives and integrates diverse input signals, and drives Cut expression with high temporospatial precision. Secondly, Cut, as a homeobox transcription factor, may intrinsically possess the ability to specify and confer organ or tissue identities, analogous to the classic homeotic genes such as Antennapedia, by simultaneously erasing old cell identities and writing new ones.We reason that Cut might be particularly suitable for being a master cell identity switch in diverse biological contexts. Firstly, Drosophila ovary Interestingly, our results revealed that the spatial and temporal cues might intersect by an unexpected ‘poising and bridging’ mechanism to dictate the midgut-to-renal progenitor identity switch . In thisAlthough long-range chromatin looping has been found at numerous gene loci , the ideDrosophila stocks used in this study include: UAS-Cut ); FRT19A, C145cut ; UAS-Wg-HA ; UAS-Arm-S10 ; UAS-cut-RNAi ; UAS-br-RNAi ; esg-Gal4, tubP-Gal80ts, UAS-GFP  ; Rab3-GFP ; UAS-CD8-RFP ; cut-intron2-Gal4 ; NRE-GFP ; UAS-Sgg.S9A ; UAS-EcR-DN ; UAS-wg-RNAi ; UAS-Br-Z1 ; UAS-Br-Z3 ; UAS-Br-Z4 ; FRT19A, 3dsh ; UAS-TCF ; UAS-Nipped-B-RNAi ; UAS-SA-RNAi ; UAS-Eip74EF-RNAi ; UAS-Eip75B ; UAS-white-RNAi ; Act5C-FRT>stop>FRT-lacZ.nls .Fly culcture and crosses were performed according to standard procedures.  UAS-GFP ; wg-Gal4 ; UAS-TCFwg-Gal4 transgenic flies were generated by integrating the Gal4 containing plasmid RIVGal4  from fz three reporter Fw: 5’-GAACGAAAGAGTTGGCAGAGAG−3’; fz three reporter Rv: 5’-GCTTAGTGGGTTTCAGGAGG−3’; ct reporter Fw: 5’-GCCGAGATGCGGTAGTAAAACG−3’; ct reporter Rv: 5’-CTGTTTGTTTCTGGCGAGCTTA −3’.The PCR primers are listed as below: To generate UAS-FLAG-Br-Z2-FL; UAS-FLAG-Br-Z2-ΔBTB; UAS-FLAG-Br-Z2-Δ5AA or UAS-FLAG-Br-Z2-ΔZF transgenic lines, full length or truncated version of FLAG-Br-Z2 was inserted into pUAST vector, followed by phiC31-mediated integration into the attP2 landing site.For Coimmunoprecipitation experiments, Arm-HA in pcDNA3.1 (Invitrogen) was made by cloning Arm-S10 from genome DNA extracted from UAS-Arm-S10 transgenic fly line , followed by replacing Myc tag with HA tag (YPYDVPDYA). FLAG-tagged Br isoforms in pcDNA3.1 were constructed by cloning cDNA of each isoform from corresponding br isoform-specific transgenic lines using genomic DNA PCR. aa 191–514; aa 1–190; aa 32–97, aa 439–492 and aa 46–50 of Br-Z2 were deleted to make Br-Z2-N, Br-Z2-C, Br-Z2-ΔBTB, Br-Z2-ΔZF and Br-Z2-Δ5AA respectively. pCMV-Myc-TCF is a generous gift from Dr. Esther M Verheyen.To generate MARCM clones shown in To generate MARCM clones shown in To generate MARCM clones shown in esg-Gal4, Gal80ts, UAS-CD8-GFP; UAS-w-RNAi or esg-Gal4, Gal80ts, UAS-CD8-GFP; UAS-cut-RNAi flies with UAS-FLP; Act5C-FRT>stop>FRT-lacZ.nls flies. Embryos were collected and kept at 18°C. Late third instar larvae were shifted to 29°C until dissection at early adult stage (2–3 days after eclosion).The lineage tracing experiments as shown in For intestine-renal tubule immunostaining, samples were dissected in Schneider’s insect medium (Sigma-Aldrich) and proceeded as previously described  with modPrimary antibodies used in this study were chicken anti-GFP , mouse anti-Pros , mouse anti-Cut (2B10) , mouse anti-Broad-core (25E9) , mouse anti-Nc82 , mouse anti-AstA , rabbit anti-Pdm1 , mouse anti-β-galactosidase , rabbit anti-β-galactosidase , rabbit anti-phospho-Histone H3 .CRL-3216 obtained from Dr. Hong Wu’s laboratory, Peking University, and authenticated by ATCC) were maintained in DMEM medium (Invitrogen) supplemented with 10% FBS at 37 ˚C and 5% CO2. DNA transfection was performed using a standard polyethylenimine (PEI) protocol.Human embryonic kidney HEK293T cells  cells  were cultured at 25°C in Schneider’s Drosophila medium Drosophila Medium (Invitrogen) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. DNA transfection of S2 cells were carried out using Effectene Transfection Reagent (QIAGEN) according to manufacturer’s protocol.Both cell lines used in this study have been tested for and confirmed to be negative for mycoplasma contamination, using short tandem repeat (STR) profiling technique.3VO4] and kept on ice for 20 min. Cell extracts were sonicated with Bioruptor Plus (Biosense) at 4°C with low power for 5 cycles of 10 s on/10 s off. The cell extracts were clarified by centrifugation, and proteins immobilized by binding to anti-FLAG M2 (Sigma-Aldrich) affinity gel for 4 hr at 4°C. Beads were washed and proteins recovered directly in SDS-PAGE sample buffer. Rabbit anti-Flag , Rabbit anti-c-myc  or rabbit anti-HA (Santa Cruz Biotechnologies) were used for western blot analysis.Coimmunoprecipitation assays in HEK293T cell extracts were performed as previously described . BrieflyIsoform-specific rabbit anti-Br-Z2 antibody was generated in this study  and used at 1:40 for immunostaining.The DNA-binding domains (DBD) of TCF (aa 271–408) and Br-Z2 (aa 432–514) were cloned into pGEX-6P-1. GST-tagged protein was purified by ProteinIso GST resin (Transgen Biotech) through column buffer (25 mM HEPES (pH 7.6), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 10 mM β-mercaptoethanol, 2 mM PMSF). After washing, protein was eluted with 100 mM glutathione in column buffer. Protein concentrations were measured by Coomassie stained gels. Known concentrations of BSA  were used as a standard.2, 0.1 mg/ml BSA, 50 ug/ml poly(dI-dC), 5% glycerol) for 45 min. The reaction mixture was loaded and resolved in 8% TBE gel. Amounts of recombinant protein used per reaction were as follows: 1.2–7.2 ug GST-TCF-DBD (E probe); 0.01–0.2 ug GST-Br-Z2-DBD (P probe) and 0.2–1 ug GST-Br-Z2-DBD (E1 probe).Increasing concentration of purified GST-tagged protein and 10 fmol biotin-labelled double-stranded DNA substrate were incubated at 25°C in 20 μl reaction buffer (20 mM HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 2 mM DTT, 6 mM MgClDrosophila S2 cells according to manufacturer’s instructions . Briefly, after transfection and fixation, S2 cells were incubated with primary antibodies at RT for 3 hr, followed by incubation with Duolink PLA probes (1:12) at 37°C for 1 hr, ligation at 37°C for 1 hr and amplification at 37°C for 2 hr. Primary antibodies used were rabbit anti-Myc (1:200) and mouse-anti-Br-core (1:150).Duolink in situ PLA was performed with Length of ureter was measured with Leica Application Suite 2.6.3 from Leica Microsystems. For quantification of the intensity of antibody staining, images were taken with the same confocal settings and the mean fluorescence intensity was measured with Histogram function of Adobe Photoshop. Unpaired Student’s t-tests were used for statistical analysis between two groups. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.cut via chromatin looping dictates natural lineage reprogramming" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Temporospatial induction of homeodomain gene The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Xu and colleagues make an important contribution on a form of cell identity reprogramming that depends on the intersection of spatial information dictated by Wg signaling and a temporal cue downstream of ecdysone signaling during metamorphosis. Intersection of these cues leads to induction of the Cut homeodomain transcription factor and conversion of midgut progenitors to a renal progenitor identity. In addition to the insight into a mechanism of temporospatial control of cell identity specification, insights gained here regarding factors that control transcription factor expression may be broadly applicable to tissue reprogramming and stem cell biology.Drosophila. Cut and related genes had already been implicated in kidney development. Spatiotemporal regulation of the cut gene is now described here, where during metamorphosis some Esg+ progenitor cells migrate from the midgut to the renal tubules where they express Cut to give rise to renal progenitor cells.Through immunofluorescence, the authors describe the molecular regulation of, and the role of, the cut gene during midgut to renal lineage conversion in Immunofluorescent analyses found that Cut is not necessary for midgut to renal tubule cell migration. The loss of Cut was however lethal to the majority of flies. Escaper flies lacked the lower ureter region and gave rise to intestinal Pros+ Cut- EE cells in the renal tubules. These anomalies suggest that Cut modulates a key renal developmental step by acting as a master identity switch. Cut expression in APF cells near the midgut-hindgut boundary is minimally regulated by Wg and ecdysone signalling. Further experiments identify a novel enhancer element and a promoter-enhancer looping model is proposed.Drosophila and makes use of robust genetic and cellular tools. These results alone may warrant publication.The role of Cut throughout fly development is extensively shown. This part of the proposed model is supported by extensive images of developing Essential revisions:Specific important comments that need to be addressed.1) The data to support the conclusion in the paper are strong, but an exception is the data to support a Broad-dependent promoter-enhancer looping mechanism. Although perfectly plausible (and even likely), the looping model is not well supported by the experiments. In order to support this idea to the extent that the authors claim, we think additional experiments would be required, such as a more direct experimental measure of the ability of Broad to loop DNA to pin down this proposed mechanism. If these experiments are not speedily feasible the conclusion on this looping mechanism should be alluded to in the Discussion section but removed from the Title and not presented in the text as bona fide.2) Other experiments  are short on detail and this part should be addressed.eLife.3) Another substantial difficulty was that the inferences regarding developmental transitions that occur at the onset of metamorphosis. These were often difficult to follow and not written in a way that is easily grasped by non-experts. The same is true for the G-TRACE results. The authors run through these data fairly rapidly without making interim conclusions or putting their observations into context. There is a lot for the reader to keep track of cell name, cell position in the different organs, location of different organs and organ parts, ploidy, cut expression status, Esg status, developmental stage, and potentially unfamiliar acronyms build up quickly. Since this description sets up the remainder of the paper, it is important to rewrite these sections with a general reader in mind to help increase the appeal to the readership of  Essential revisions:Specific important comments that need to be addressed.1) The data to support the conclusion in the paper are strong, but an exception is the data to support a Broad-dependent promoter-enhancer looping mechanism. Although perfectly plausible (and even likely), the looping model is not well supported by the experiments. In order to support this idea to the extent that the authors claim, we think additional experiments would be required, such as a more direct experimental measure of the ability of Broad to loop DNA to pin down this proposed mechanism. If these experiments are not speedily feasible the conclusion on this looping mechanism should be alluded to in the Discussion section but removed from the Title and not presented in the text as bona fide.We wish to thank the editors and reviewers for all the efforts they have put into reviewing and improving our manuscript. We agree with the reviewers that more direct evidence supporting the Broad-dependent-looping model, such as a 3C (chromatin confirmation capture) experiment using FACS-purified future renal progenitors at P0 stage, would be ideal. This is indeed some experiment that we have been thinking about as well. However, due to the small number of future renal progenitors in each fly pupal midgut, it would be too time-consuming and technically challenging to complete such 3C experiment in a timely manner. Therefore, we decided to follow reviewers’ insightful suggestions and tone down the language on this looping mechanism. The manuscript has been modified as following: (1) remove “via chromatin looping” from the title; (2) move the working model of cut enhancer-promoter looping to the Discussion section; (3) tone down our statements on this looping model throughout the text. As an example, “Our results therefore led us to propose an unexpected poising-and-bridging mechanism whereby spatial and temporal cues intersect via chromatin looping to turn on a master transcription factor and dictate efficient and precise lineage reprogramming” has been changed to “Our results therefore led us to propose an unexpected poising-and-bridging mechanism whereby spatial and temporal cues intersect, likely via chromatin looping, to turn on a master transcription factor and dictate efficient and precise lineage reprogramming” (Abstract).2) Other experiments  are short on detail and this part should be addressed.Thank you for this important point. For the RNAi screen, we have now added one paragraph describing the strategy and detailed procedure of this genome-wide RNAi-based screen , as well as a supplemental figure panel  demonstrating the cut-RNAi-induced renal tubule phenotype that we initially observed while carrying out the screen.As for the identification of the new cut enhancer element, we have now added a few sentences describing how the new enhancer was found: “Out of 22 cut enhancer-Gal4 driver lines from the Janelia Gal4 collection, in which Gal4 is expressed under the control of cut enhancer fragments  , we identified one line, R35B08-Gal4, which drove UAS-CD8-RFP expression specifically in future RPs at metamorphosis ”. We also added one new figure panel  demonstrating that CD8-RFP driven by this newly-identified cut enhancer-Gal4 colocalizes with endogenous Cut in future renal progenitors at the onset of metamorphosis.Furthermore, to provide more detailed information regarding how we decided the size and sequence of the EMSA probes, we have now added the consensus TCF-binding site, helper site and Br-Z2-binding site to the figure legend of Figure 7—figure supplement 2.3) Another substantial difficulty was that the inferences regarding developmental transitions that occur at the onset of metamorphosis. These were often difficult to follow and not written in a way that is easily grasped by non-experts. The same is true for the G-TRACE results. The authors run through these data fairly rapidly without making interim conclusions or putting their observations into context. There is a lot for the reader to keep track of cell name, cell position in the different organs, location of different organs and organ parts, ploidy, cut expression status, Esg status, developmental stage, and potentially unfamiliar acronyms build up quickly. Since this description sets up the remainder of the paper, it is important to rewrite these sections with a general reader in mind to help increase the appeal to the readership of eLife.eLife, we have now carefully labelled the organ parts, such as midgut, hindgut, lower ureter, upper ureter and lower tubule, in most panels of main Figure 1 and 2.We thank the reviewers for this constructive suggestion. Since fly renal tubule is not a widely-used model system, its anatomy, cell composition and dynamic changes throughout developmental stages could cause potential difficulties for general readers. Therefore, to help increase the appeal of our manuscript to the broad readership of + progenitors and all their subsequent daughter cells . Meanwhile, GFP reveals the real-time expression of esg-Gal4 ” (subsection “RPs displayed MP characteristics upon Cut depletion”). Furthermore, in light of reviewers’ comments, we have now added an interim conclusion “These results strongly suggested that, upon cut depletion, Esg+ progenitors migrate normally onto the renal tubules, yet fail to switch into renal identity and differentiate into midgut cells along renal tubules” (subsection “RPs displayed MP characteristics upon Cut depletion”).As for the G-TRACE experiment, we have now added two new figure panels to show the genetic schema  and experimental timeline  of this lineage-tracing experiment. Furthermore, we included detailed description of the lineage-tracing strategy “We used temperature-sensitive esg-Gal4, UAS-CD8-GFP, tub-Gal80ts system to drive FLP (flippase) expression, transferred animals from permissive temperature (18°C) to restrictive temperature (29°C) at late 3rd instar larval stage and analyzed the renal phenotypes at early adult stages . Upon esg-Gal4-driven expression of FLP recombinase, a transcriptional stop cassette flanked by FRT sites is excised, resulting in lacZ expression in Esg


A recent developmental study of gene expression by Espinosa-Medina, Brunet and colleagues sparked controversy by asserting a revised nomenclature for divisions of the autonomic motor system. Should we re-classify the sacral autonomic outflow as sympathetic, as now suggested, or does it rightly belong to the parasympathetic system, as defined by Langley nearly 100 years ago? Arguments for rejecting Espinosa-Medina, Brunet et al.’s scheme subsequently appeared in e-letters and brief reviews. A more recent commentary in this journal by Brunet and colleagues responded to these criticisms by labeling Langley’s scheme as a historical myth perpetuated by ignorance. In reaction to this heated exchange, I now examine both sides to the controversy, together with purported errors by the pioneers in the field. I then explain, once more, why the sacral outflow should remain known as parasympathetic, and outline suggestions for future experimentation to advance the understanding of cellular identity in the autonomic motor system. The principles drawn from autonomic neuroscience are essential for understanding human physiology and the pathology of disease . Due to Phox2a, Tbx20, Tbx2 and Tbx3 in nX, but not in the thoracic or sacral cord. Conversely, Foxp1 was detected in the thoracic and sacral cord, but not in nX. Based on the co-segregation of thoracic and sacral traits from cranial traits, it was concluded that thoracic and sacral pools of preganglionic neurons share a common sympathetic identity. Alternatively, another interpretation is possible. Thoracic and sacral preganglionic neurons may simply share a common spinal identity [Hmx2 and Hmx3 were detected in several cranial parasympathetic ganglia, but not in lumbar paravertebral sympathetic ganglia or in the pelvic ganglion. Conversely, the sympathetic ganglia and pelvic ganglion selectively express Islet1, Gata3 and Hand1. In addition, genetic deletion of Olig2, which disrupts formation of cranial parasympathetic ganglia [The key arguments that support the Brunet conjecture rest on the differential expression of transcription factors in mice, primarily on embryonic day 13.5 . By compidentity . The samidentity . Hmx2 an ganglia , 8, fail ganglia , it is aFin de siècle neuroscience, Langley’s insight appears more akin to the imaginative ideas developed by Santiago Ramon y Cajal during his ground-breaking explorations of neuroanatomy. We can only hope that in 100 years, future neuroscientists will find something of enduring value in our early 21st century efforts, crude as they may be! Despite these cautions, I agree with the Brunet group that one should acknowledge the history of ideas as a prelude to incorporating genetic mechanisms into autonomic neuroscience.The Brunet group’s recent commentary  re-asserTo recount the history of autonomic neuroscience, one must acknowledge John Newport Langley 1852–1925) , 16. Bui25 , 16. “Sympathetic nerves have no special relation to sympathies.” page 7 Langley opens by recognizing Jacques-Benigne Winslow , an inflLangley introduced the concept of an “autonomic” nervous system in order to distinguish nerves that control smooth muscles and glands from the somatic motor nerves that control striated muscles. In choosing the term autonomic, Langley also sought to find a better word than “vegetative” and “involuntary”. He argued that vegetative implied a false relationship with plants and that involuntary was inadequate because people can initiate certain autonomic actions as a matter of will . Despite this rationale, use of the term vegetative persists in some non-English speaking countries .“…the chief objection to calling the whole autonomic system sympathetic is that it confuses instead of simplifying nomenclature:” page 7 .The decision to move beyond Winslow’s earlier usage by dividing the autonomic system into three divisions was essential in order to capture its organization and function.Ironically, the same logic applies today to Brunet’s conjecture. Reverting to a common name for the thoraco-lumbar and sacral autonomic outflows does not simplify discussion or understanding of the autonomic motor system because these elements display distinct features , 33.Langley defined the sympathetic, parasympathetic and enteric systems using several criteria , beginniToday’s controversy, like most of Langley’s monograph  focuses “The facts that the sympathetic innervated the whole body, whilst the cranial and sacral outflows innervated parts only, and that the sympathetic had, in general, opposite functional effects from those of the other autonomic nerves, indicated that the sympathetic was distinct from the rest.” page 8 .The sympathetic and parasympathetic outflows differ in terms of their targets, the pathways of their peripheral nerves and their functional attributes. One can trace these concepts to Langley, together with the idea of functional opposition—they all remain deeply embedded as core principles of autonomic neuroscience.In general, the sympathetic system, but not the parasympathetic system , innervates the skin. An exception to this pattern has been reported in the lower lip of the cat , 22.Thermoregulation is exclusively sympathetic and arises primarily through thoraco-lumbar control of the cutaneous circulation, piloerection, sweat glands and brown fat.Blood pressure control operates primarily through sympathetic regulation of the cardiovascular system and kidneys.Vagal parasympathetic inhibition of the heart opposes sympathetic excitation.Sacral parasympathetic activation promotes micturition, while sympathetic activation promotes urine retention , 5.Sympathetic neurons in paravertebral chain ganglia are selectively innervated by thoraco-lumbar sympathetic preganglionic neurons, and not by sacral or suprasegmental parasympathetic preganglionic neurons. The axons of sacral preganglionic neurons never enter the paravertebral sympathetic chain.Sympathetic ganglia  are intimately associated with large arteries, while parasympathetic ganglia are often embedded within target organs . Brunet’s group takes issue with this interpretation , noting The following examples support Langley’s view, but are not explained by the Brunet conjecture.“The only structures markedly influenced by sympathetic stimulation which are not influenced by adrenaline after nerve section are the sweat glands of the cat and some other mammals.” page 29 The recent papers from Brunet’s group , 10 corrBy the 1930s it became clear that cholinergic sympathetic mediation of sweating was an exception to the rule that postganglionic sympathetic neurons use norepinephrine as their transmitter . The firThe work from Professor Brunet’s laboratory serves an important purpose by illustrating the power of developmental molecular genetics to illuminate important features of the autonomic motor system. It should motivate us to re-examine long-held beliefs. Although the interesting results from their experiments do not justify a reclassification of the sacral parasympathetic as sympathetic, they point to a path forward. For the time being, I conclude that Langley got it right concerning the three divisions of the autonomic motor system. Moving forward, transcriptomic methods now make it possible to identify patterns of gene expression that characterize distinctions between populations of adult autonomic neurons . This ap


In human cells, DNA is hierarchically organized and assembled with histones and DNA-binding proteins in three dimensions. Chromatin interactions play important roles in genome architecture and gene regulation, including robustness in the developmental stages and flexibility during the cell cycle. Here we propose in situ Hi-C method named Bridge Linker-Hi-C (BL-Hi-C) for capturing structural and regulatory chromatin interactions by restriction enzyme targeting and two-step proximity ligation. This method improves the sensitivity and specificity of active chromatin loop detection and can reveal the regulatory enhancer-promoter architecture better than conventional methods at a lower sequencing depth and with a simpler protocol. We demonstrate its utility with two well-studied developmental loci: the beta-globin and HOXC cluster regions. Chromatin interactions and genome architecture are key regulators of gene expression. Here the authors present Bridge-Linker-Hi-C to map active chromatin loops and enhancer-promoter interactions. The chromatin architecture is established during early development in mammals and remains dynamic during the cell cycle on the scale of topologically associated domains and compartments5. A growing body of evidence has shown that architectural proteins and transcription factors play important roles in maintaining distal chromatin interactions and regulating the chromatin conformation on a much smaller scale9. To directly investigate this sophisticated structure, several variations of 3D genomics methods have been published18. For example, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and HiChIP utilize the chromatin immunoprecipitation (ChIP) assay to enrich specific protein-maintained chromatin contacts in millions of cells18, and Capture Hi-C (CHi-C) enriches chromatin interactions based on known and pre-specified genomic regions19. A further simplified approach to interrogating the genome-wide chromatin architecture at high resolution without additional antibody enrichment or bait fragment capture would be highly desirable. This goal led to our BL-Hi-C method, which provides a new, to the best of our knowledge, strategy for the enrichment of protein-centric chromatin contacts.High-throughput chromosome conformation capture (Hi-C) assays significantly advanced our understanding of three-dimensional (3D)-genome organization, i.e., DNA hierarchically folded into chromatin fibers, domains, and compartmentsHaeIII, a four-base cutter that recognizes “GGCC”, is used for digestion. Theoretically, HaeIII cuts the human genome every 342 bp on average, which is similar to the cutting frequency of MboI, which is every 401 bp on average. However, considering that DNA is occupied by structural and regulatory proteins associated with the genome in the nucleus, we found that HaeIII theoretically targets active regions  more closely than other enzymes used previously  and compared the results with recently published in situ Hi-C and HiChIP dataods Fig. . The rat23. When we examined CTCF and RNAPII ChIP-seq peaks in chromatin interaction anchor regions, there were ~ 1.3–3.3-fold CTCF enrichment and ~ 2.0–5.4-fold RNAPII enrichment for BL-Hi-C PETs compared to in situ Hi-C and HiChIP  and RNA polymerase II (RNAPII) in regulating the genome architecture and enhancer-promoter interactionshIP Fig. . Furtherons Fig. . Notablyig. Fig. . Furtherig. Fig. . We examons Fig. . Taken t21. Here we identified 10 014 loops from 639 M reads by BL-Hi-C, which is much more efficient than in situ Hi-C, which identified 6057 loops from 1.37 B reads. We further grouped the loops into common loops detected by both methods and specific loops detected only by BL-Hi-C or only by in situ Hi-C  than did in situ Hi-C. Moreover, we observed similar results in another well-known active region, HOXC  provided by the method, the key advantages of BL-Hi-C are as follows: the 4-bp CG-rich cutting enzyme, which can digest shorter restriction fragments from CTCF or active transcription factor-associated chromatin loops, increasing the re-ligation probability for the fragments bound by specific protein complexes2 atmosphere. The maximal density of K562 cells was no more than 700K per ml, and the culture was split to obtain a density of 400K per ml when passaging cells.K562 chronic myelogenous leukemia cells were cultured in Roswell Park Memorial Institute 1640  medium supplemented with 10% fetal bovine serum (Life Technologies) at 37 °C in a 5% COHaeIII and two-step ligation includes cell crosslinking, cell lysis, restriction digestion (steps a–d), proximity ligation (steps a–c), DNA purification, sonication, library construction, PCR amplification, and sequencing. The enzyme-modified BL-Hi-C assay for the enzyme MboI, HindIII, or others with overhangs and two-step ligation includes cell crosslinking, cell lysis, restriction digestion , proximity ligation (steps a–c), DNA purification, sonication, library construction, PCR amplification, and sequencing. The ligation-modified BL-Hi-C assay for the enzyme HaeIII and one-step ligation includes cell crosslinking, cell lysis, restriction digestion (steps a–d), proximity ligation (steps d–j), DNA purification, sonication, library construction, PCR amplification, and sequencing.All the BL-Hi-C data were generated using the step-by-step protocols supplied in the Supplementary Methods. The standard BL-Hi-C assay for the enzyme The standard 4C-seq protocol utilized double-enzyme digestion and circulation for primer targeting on the bait regions. Here we targeted primers to the bait regions and Y-type adaptor of BL-Hi-C libraries, generating genome-wide interactions with the bait regions. Thus, libraries were prepared by cell culture, crosslinking, cell lysis, digestion, DNA purification, sonication, enrichment, and library construction procedures similar to those in the BL-Hi-C protocol. Then, two rounds of PCR were performed. The 4C viewpoint primer  and I7 adaptor (GTGACTGGAGTTCAGACGTGT) were used for the first round of PCR. After the DNA products were purified with AMPure XP beads, I5 (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) with the 4C viewpoint sequences, including anchor 1 to anchor 3, and I7 (CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGT) with barcodes were used for the second round. Then, the libraries were sequenced on an Illumina sequencer Hiseq X Ten.We used ChIA-PET2 software to process the BL-Hi-C sequencing data, including linker trimming, read alignment (BWA), PET formation, and duplicate removal. The PETs were further adapted to enable the software homer to generate contact matrixes and heatmaps, to allow the software ChIA-PET2 or MICC to perform peak calling or PET clustering, among other functions.The 4C-seq data were processed by ChIA-PET2 software to obtain PET files and further PET clusters with interaction counts using the following command: -m 1 -t 4 -k 2 -e 1 -l 15 -S 500 -A ACGCGATATCTTATC -B AGTCAGATAAGATAT -M “--nomodel -q 0.05 -B --SPMR --call-summits”. Then, the designed bait sequences for the enhancer anchor were used to select the target PETs and clusters for each 4C-seq library, which were further visualized by the WashU Epigenome Browser.et al.), the public CTCF and RNAPII ChIP-seq data on the K562 cell line were used. For HiChIP, the public ChIP-seq data on the GM12878 cell line were used. For in situ Hi-C , the public ChIP-seq data on the H1hesc cell line were used. A similar strategy was applied for the overlap of ChromHMM annotation. ENCODE processed the “bam” files for the input, and the overlapping from the CTCF and RNAPII ChIP-seq data was used to show the enrichment pattern. Then, the bedtools command “bedtools coverage -sorted” was applied to calculate the depth for each group of CTCF or RNAPII peaks. In addition, the homer software command “annotatePeaks.pl” was used to calculate the enrichment of genomic features for each group.We converted the PETs to bed files for both the BL-Hi-C and public data for further enrichment analysis, or the ChIA-PET2 output file “rmdup.bedpe.tag” was used directly. Then, we used the bedtools software command “bedtools intersect -u” to identify the PETs that overlapped with the public ChIP-seq peaks. For BL-Hi-C and in situ Hi-C  under accession number GSE93921. All other relevant data are available from the corresponding author on request.Supplementary InformationPeer Review File


AOX1) promoter is the most popular but strictly-regulated methanol inducible promoter for heterologous protein expression in Pichia pastoris. In recent years, AOX1 promoter libraries have been developed with deletion or insertion methods. The present research manipulated poly (dA:dT) tracts in this promoter to control promoter strength, which hadn’t been tried before. There were 34 variants derived from the native AOX1 promoter constructed. And variants were integrated into the same genomic location and upstream of the same reporter gene porcine growth hormone (pGH). To test the transferability of the results obtained from reporter gene pGH, the variants were connected to reporter gene Lac Z. The resulted promoter library spanned an activity range between 0.25 and 3.5 fold of the wild-type promoter activity. In addition, activities of variants correlated with their predicted nucleosome architecture, which were directed by poly (dA:dT) tracts. The cumulative sum of predicted nucleosome affinity across the region (−820 to −540) was related to promoters strength in single deletion variants on a proportional basis. Overall, the research promotes understanding of the regulatory patterns for AOX1 promoter and suggested that varying promoter expression of engineering nucleosome architecture was also a feasible approach in P. pastoris.Alcohol oxidase I ( Pichia pastoris (Komagataella phaffi) has been commonly used for production of heterologous protein2. Alcohol oxidase I (AOX1) promoter, remarkably strong and tightly regulated by methanol4, turns out to be most popular. Though with favorable properties and industrial importance, AOX1 promoter lacks further study, considering the fact that its transcriptional regulatory mechanism has not been clarified3. Fine-tuning of heterologous gene expression is necessary for maximization of protein expression level. And developing an efficient strategy for optimizing promoter activity should be based on a better understanding of regulatory mechanism of promoter.The methylotrophic yeast AOX1 promoter have focused on deletion and duplication of putative transcription factor-binding sites and identification of upstream activation sequence6. Although those traditional methods have proved successful in creating promoter library, the resulted variants activities were ~1.6 fold of wild-type AOX1 promoter activity5. There have been great efforts made to study rational design of AOX1 core promoter in recent years. For instance, Thomas Vogl et al.3 designed AOX1 core promoter by using a consensus sequence of natural core promoters and common transcription factor binding site motifs. And the activities of synthetic variants ranged from 10% to 117% of wild-type AOX1 promoter. Besides, Portela RM et al.4 designed 112 synthetic promoters based on sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs and fused synthetic core promoters to AOX1cis-regulatory modules (CRMs). These studies provided new idea for engineering promoters including AOX1 promoter.Previous studies of optimizing 8. These sequences harbor a shorter helical structure and a narrow minor groove, which serves to resist the bending required for histone binding9. Poly (dA:dT) tracts could create a “barrier” that favors the formation of highly positioned nucleosomes adjacent to these tracts, which in turn directs the positions of neighboring nucleosomes10. In this sense, these tracts could exert influence on nucleosome occupancy and affinity. Many studies show that poly (dA:dT) tracts are important for transcriptional regulation12. Due to their unusual structure, nucleosomes are strongly depleted from these tracts and their own flanking sequence. Moreover, narrowing nucleosome occupancy over the region in vicinity of those tracts can increase the accessibility of DNA in the same region to transcriptional factor binding sites7. Poly (dA:dT) tracts have been used to regulate promoters in yeast. Altering the presence and length of native poly (dA:dT) tracts can increase accessibility to the nearby transcription factor-binding sites which are covered by nucleosomes, thus regulating promoter activities14.Poly (dA:dT) tracts-homopolymeric stretches of deoxyadenosine are highly abundant in yeast genomeAOX1 promoter. Therefore, the present research aims to analyze the transcriptional effect of poly (dA:dT) tracts in AOX1 promoter by altering the presence and length of native poly (dA:dT) element in different sites. This study indicates that deletion or lengthening native poly (dA:dT) tracts can alter the variants activities ranging from ~0.25 to ~3.5 fold of wild-type promoter activity, proving that the method is more effective than traditional methods for improving AOX1 promoter activities. In addition, there is also prediction on occupancy and affinity of nucleosomes. And research shows that occupancy and affinity of nucleosomes in certain regions of AOX1 promoter are correlated with promoter activities.Poly (dA:dT) tracts are highly prevalent in yeast promoters including 14. In order to study the influence of poly (dA:dT) on transcription, there were 34 variants constructed on the basis of the native Pichia pastoris AOX1 promoter. Eight perfect poly (dA:dT) tracts and imperfect tracts  tracts in eukaryotic genome, especially in promoters. Manipulation of poly (dA:dT) tracts can cause changes of nucleosome organization, thus altering the promoter strengthGAP promoter locus) in all strains. The copy numbers of reporter gene, pGH and Lac Z in transformants were confirmed by qPCR  and Lac Z respectively Table . The resCR Table . FinallyAOX1 promoter based on the hidden Markov model, of which practicability was validated by previous work15. Providing the fact that the hidden Markov model is usually used for predicting genome nucleosome occupancy, the prediction is able to reflect nucleosome occupancy of AOX1 promoter in repressed state.In order to study correlation between nucleosome structure and poly (dA:dT) tracts, there is prediction of nucleosome occupancy of AOX1 promoter  tracts of most of variants has slight influence on nucleosome positioning. But there is noticeable increase in the predicted affinity of nucleosomes which cover correspondent poly (dA:dT) tracts Fig.  and b. Dter Fig. . Howeverter Fig. .After addition of 15 bp poly (dA:dT) tracts in different sites, there was a reduction in nucleosome occupancy upon addition of a nearby tract. The nucleosomes  positioning of Add-S1 and Add-S3 variants had been influenced significantly. And the gap between nucleosome (−4) and nucleosome (−3) increased by nearly 90 base pairs, which influences the position of nucleosome (−5) and (−4) Fig. . In the AOX1 promoter activity, there are series of variants containing different length of poly (dA:dT) tracts  tract in different sites respectively. Expression levels of pGH under variants  increased to 1.5~2 fold of WT  tracts in two sites. As a result, there was a considerable rise in the strength (2~3.5 fold of WT) of variants . However, double deletion in other sites caused no change in the promoter activity  increased to 1.2~1.5 fold of WT while that of variants  dropped  tract is a major factor influencing nucleosome positioning. In order to test the influence of poly dA:dT on  WT Fig. . The var WT Fig. . In an aity Fig. . For theped Fig. . Howeverped Fig. , the actPCR Fig. . There wPCR Fig. .Figure 2pGH  –tract-deleted variants were higher than that of promoters with inserting 15 bp poly (dA:dT) tracts.To test the transferability of research results of reporter gene and pGH, the variants were connected to reporter gene and Lac Z. And the transcription levels of beta-galactosidase were also performed Fig. . It is fpGH Fig. . Taken tAOX1 expression could strongly restrict glucose/glycerol, the strength of wild-type and variant AOX1 promoters in the presence of glycerol by beta-galactosidase activities were also measured here. The activities of beta-galactosidase increased to 1.5~2.5 fold of WT apart from variants   Fig. . And the56) Fig. . AnalysiGiven that the transcriptional activity could be influenced by changes in poly (dA:dT) tracts, the following part probes into whether influence could be explained through prediction of nucleosome architecture. And the prediction is carried out by manipulating poly (dA:dT) tracts. There was a correlation analysis of relationship between predicted nucleosome architecture and expression level. Nucleosome architectures of Variant Del-S4, Variant Double-Del-S24 and Variant Double-Del-S34 show great similarity. But their nucleosome architectures are significantly different from that of other poly (dA:dT) –tract-deleted variants and WT variants Fig. . The nucAOX1) promoter is the most commonly used and tightly regulated metha2nol inducible promoter for heterologous protein expression in Pichia pastoris4. In the present research, a AOX1 promoter library consisting of 34 variants was connected to porcine growth hormone (pGH). And poly (dA:dT) tracts were adjusted, leading to a broad range of activities (from 0.25~3.5 fold of WT). The research indicates that systemic adjustment on poly (dA:dT) tracts in yeast promoters can contribute to create promoters which provide expression levels of a wide range.Alcohol oxidase I . However, previous studies, which were based on stochastic series with finite states, failed to figure out factors influencing AOX1 promoter activity. In the present research, systematic adjustment on poly (dA:dT) tracts is not only suitable for AOX1 but also useful for other yeast promoters, which can lay foundation for research of engineering promoter other promoters as well as AOX1 in the future.Recently, there have been various studies focusing on rational design of 14. However, the present study provides findings contrary to that of previous research of the influence of poly (dA:dT) tracts in AOX1 promoter on transcription. Deletion of these tracts in AOX1 promoter could stimulate expression to some extents while addition of 15 bp poly (dA:dT) tracts in different sites resulted in a reduction in expression level especially in double sites addition variants.Some studies showed that architecture of nucleosome surrounding transcription factor-binding sites could be an important factor influencing the strength of yeast promoters. Nuclesome architecture plays a leading role in defining yeast promoter activity, which makes it able to design synthetic promoters. Poly (dA:dT) tracts not only disturb nucleosome formation but also increase accessibility of transcription factor-binding sites14 nearby, thus promoting expression. Some research indicated that the properties and spatial arrangement of poly (dA:dT) tracts could exert considerable impact on transcriptionAOX1 promoter. Many studies focused on the regulatory role of poly (dA:dT) tracts in simple constitutive promoter such as HIS3 promoter14. However, there has not any reported research centering on the regulatory role of poly (dA:dT) tracts in inducible promoters including AOX1 promoter so far. Providing the fact that AOX1 promoter is regulated by methanol, the regulation by poly (dA:dT) tracts on AOX1 promoter would be more complex than that on constitutive promoter. According to Fig. 5 study, compared to WT, deletion of d1 (−777 to −712) or Rap (−615 to 601) resulted in higher promoter activity, suggesting that this region contains repressive binding site5. Despite, with methanol induction, deletion of Hsf1 (−805 to −798) and d6 (−253 to −224) resulted in decreasing promoter activities, the promoter activities in depression (carbon source was depleted) increased, suggesting that these fragments probably served as the binding site for both activators and repressors. Recently, A Cys2His2 zinc finger transcriptional regulator PpNrg1 has been identified as a repressor of AOX1 promoter16. Two binding sites of Cys2His2 zinc finger transcriptional regulator PpNrg1 contain conserved regions with CYCCNY which are binding sites of methanol expression regulator (Mxr1p) while a binding site contains a part of region of Prm1 binding sites17. To sum up, there are both of activating binding sites and repressor binding sites distributing on AOX1 promoters. And some of them even share the same region. These features suggest that competition between activators and repressors plays an important role in regulating transcription of AOX1 promoter.It is speculated that these influences could arise from some specialties of 18 on the binding sites of Mxr1p when regulation of AOX1 promoter works. Increase in predicted nucleosome affinity of these regions can lead to decrease in the binding affinity of repression factor according to the model of Raveh-Sadka’s study14. As the promoter is activated by methanol, Mxr1p will translocate to nucleus, thus regulating the promoter. Given that the main competitor for binding is histone, it would be easier for activators to bind on the Mxr1p binding site (MXRE). On the contrary, decrease in the nucleosome affinities of these regions will make Mxr1p compete with PpNrg1which has already been band on the Mxr1p sites before the translocation of Mxr1p into nucleus. This assumption makes it easier to understand the regulatory role of poly (dA:dT) tracts discussed in the study. The regions (−820 to −540) contain PpNrg1 binding sites (−796 to −785) which could also be identified by Mxr1p. Increasing the predicted affinity of nucleosome nearby these Nrg1binding sites could reduce the affinity of Nrg1 repressor. Therefore, compared with Nrg1 which has already been bound to MXRE, Mxr1 can compete with histone in an easier way. In this sense, depressing AOX1 promoter can improve the efficiency of binding DNA to regulator.PpNrg1 probably competes with activator Mxr1p6. The activity of mutant AOX1 promoter, which contains three copies of region (−638 to −510), was 1.57 fold of that of wild-type AOX1 promoter. With insertion of 15 bp poly dTs in S6 site of AOX1 promoter, the expression level of pGH increased to ~1.5 fold of wild-type , decreasing predicted nucleosome affinity could improve promoter activity Fig. . The regype Fig. , provingTheoretically, there was negligible correlation between the predicted nucleosome affinity and promoter activity in region (−585 to −185). And the research proved that predicted nucleosome affinity of extremely low level caused slight changes in promoter activity Fig. . Such in19. So the activities of Double-Add-58 and Double-Add-78 were decreased comparing with wild-type promoter.In addition, reposition of nucleosome in variants has influence in promoter activities. For example, nucleosome (−4) moved forward 50 base pairs to the upstream of promoter in variant Del-S4, variant Double-Del-24, variant Double-Del-46 and variant Double-Del-34, which resulted in location of Mxr1p/PpNrg1 binding sites  tracts, which were inserted to regions, may influence the nucleosome repositioning by remodeling complexes when chromatin was in the active state. And poly (dA:dT) tracts, which were extended, may disturb the original positioning of nucleosome when chromatin was in the active state. Therefore, most of double-addition variants showed low level expression in the present study.AOX1 promoter sequence in this study. Unprecedentedly, the present research showed that poly (dA:dT) tracts could regulate AOX1 promoter which is inducible, suggesting that varying promoter expression by engineering nucleosome architecture is also a feasible approach in P. pastoris. However, there remains a large space for study of the regulation mechanism of poly (dA:dT) tracts in AOX1 promoter.There was a promoter library created by deletion and addition of poly (dA:dT) tracts within the P. pastoris X33 (Invitrogen) was cultivated in BMGY medium in the phase of growth phase and BMMY medium in the phase of induction. There is 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen base without amino acids, 4 × 10−5% biotin, and 1% glycerol in BMGY medium. And there is 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen base without amino acids, 4 × 10% biotin, and 0.5% glycerol in BMMY medium. Escherichia coli DH5α were used for plasmid propagation while BL-21(DE3) cells were used for cloning of Lac Z coding sequence. Escherichia coli DH5α was cultivated in LB medium with temperature of 37 °C. And LB medium contains 1% tryptone, 0.5% yeast extract and 0.5% NaCl supplemented with 25 µg ml−1 Zeocin for plasmid maintenance and propagation.Strains: Plasmids: pPICZA was used for constructing mutants of AOX1 promoter while pGAPZαA (Invitrogen) was used for cloning the sequence of GAP promoter for homologous recombination with yeast genome. All plasmids used in this study are listed in Table The pGH coding sequence was obtained with the forward primer pGH-F and reverse primer pGH-R being used for clonal expansion of cDNA of porcine pituitary. After digested by EcoR I and Not I, PCR products of pGH were inserted into the vector pPICZA, thus developing pPICZA-pGH. And the expression of pGH was under control of AOX1 promoter. Sequence of GAP promoter was inserted into pPICZA-pGH, producing plasmids which were inserted into GAP promoter locus of genome with homologous recombination. As a result, a homologous region between plasmid and Pichia pastoris was created.Xba I site were used for creating a Xba I site upstream of BglII site on pPICZA-pGH by omega PCR21. In omega PCR, the two portions of the chimeric primers annealed to their complementary sites on pPICZA-pGH. The Xba I site was to be inserted into the target position of pPICZA-pGH through ~25 cycles of PCR. With processed by Dpn I, the PCR product transformed into E. coli DH5α competent cells. The pPICZA-pGH-Xba transformants were screened by a pair of primers Check-Xba-F/AOX-R.For insert GAP promoter into pPICZA-pGH, complementary chimeric primers Xba-F/Xba-R containing Bgl II and Xba I, and inserted into the vector pPICZA-pGH-Xba resulting in GAP-pPICZA-pGH.The GAP promoter fragment was amplified from plasmid pGAPZαA by PCR and digested by AOX1 (AOX194)5 and found three 5 bp perfect poly (dA:dT) tracts, and five AT rich tracts, as shown in Fig. AOX1 promoter, we performed deletion each of these tracts by deletion omega PCR21. The primers for deletion of poly (dA:dT) tracts were consist of two parts. The 5′ portion of the forward primer Deletion-S(n)-F(n = 1~8) consisted of 25 bases and was identical to the 5′-flanking sequence of the poly (dA:dT) tracts, and the 3′ portion (~25 bases) was identical to the 3′-flanking sequence of the poly (dA:dT) tracts. Thus, the poly (dA:dT) tract was removed from forward primer. The reverse primer Deletion-S(n)-R was reversed and complementary to the forward primer. In omega PCR, the deletion primers annealed to their complementary sites on wild-type AOX1 promoter of GAP-pPICZA-pGH. After treatment with Dpn I, the PCR product was transformed into E. coli DH5α competent cells. The transformants GAP-pPICZA-Del-S(n)-pGH were screened by a pair of primers Check(n)-F/AOX-R.Next, we identified several poly (dA:dT) tracts upstream the basal promoter of The construction of double sites deletion variants were based on single site deletion variants. Double deletion variant Double-Del-S12 and Double-Del-S24 were obtained by deletion of poly (dA:dT) tracts in S1 and S4 sites on single deletion variant Del-S2 respectively, and the corresponding plasmids were named GAP-pPICZA-Double-Del-S12-pGH and GAP-pPICZA-Double-Del-S24-pGH. We obtained the rest double deletion variants Double-Del-34, Double-Del-46, Double-Del-56, Double-Del-57, Double-Del-58, Double-Del-67 and Double-Del-78 by the same way. The corresponding addition variants (Add-S1~S8 and Double-Add-S12~S78) were created by the same method, except that the primers containing extra 15 bases dA/dTs. A schematic representation of plasmids for variants was shown in Fig. Lac Z coding sequence was amplified from genome of Escherichia coli BL-21(DE3) using primers LacZ-F/LacZ-R. Unfavourable EcoR I restriction sites within the Lac Z conding sequence was removed using primers LacZ-F/Del-EcoR-R. The PCR product was digested by EcoR I and Not I, and cloned into pPICZA, resulting in pPICZA-LacZ. The obtained pPICZA-LacZ was digested by EcoR I and BamH I,and the fragment containing Lac Z was used to substitute pGH in plasmids containing promoter variants. All plasmids used in this study were confirmed by DNA sequencing. All primers were listed in Table GAP promoter locus of Pichia pastoris, the nuclesome positioning and affinity of promoters were predicted by NuPoP software using Hidden Markov model15.When recombinant plasmids were inserted into P. pastoris X33 electro-competent cells were transformed with Bln I-linearized variants, using an Eppendorf Electroporator 2510  and pulsed at 1.5 KV. After electroporation, 0.5 ml of ice-cold 1 M sorbitol was added immediatedly. The suspension was transferred to a sterile 5 ml tube and added 0.5 ml of YPD  followed by incubation 1 h at 30 °C with shaking. After regeneration, aliquots were plated on YPDS  plates containing 100 μg/ml Zeocin. Positive transformants were screened by PCR using primers 5′AOX1/3′AOX1.Genomic DNA of yeast and Escherichia coli were extracted using Rapid Yeast Genomic DNA Isolation Kit and Bacterial Genomic Isolation Kit (Sangon Biotech China). The quality of genomic DNA was assessed by NanoDrop Spectrophotometer .600). The cells were centrifuged at 3,000 g for 10 min at room temperature, and then suspended in 10 ml of BMMY. Methanol was supplied in a final concentration of 0.5% every 24 h. After 48 h of induction, the pellet was collected for subsequent experiments.Colonies of transformants were cultivated in BMGY or BMMY culture medium containing 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen base without amino acids, 4 × 10–5% biotin, and 1% glycerol (BMGY) or 0.5% methanol (BMMY), respectively. Colonies of transformants were inoculated into 50 ml tube containing 10 ml of BMGY medium with 230 rpm shaking at 28 °C until the optical density reached 15 . Values for each target gene were normalized using actin. Expression values were calculated using the 2−△△Ct method22.The total RNA of yeast and porcine pituitary were extracted using total RNA Isolation Kit (Sangon Biotech China). RNA integrity was checked on 1% agarose gels and quantified using NanoDrop . After heating at 85 °C for 10 mins to denature RNA, 500 ng of total RNA was subjected to reverse transcription using the ReverTra Ace quantitative real-time PCR (qPCR) RT Kit . Stationary samples were used for real-time PCR analysis. The level of mRNA was quantified with qPCR using a commercial reagent kit. For each of the targeted genes, a pair of oligonucleotide primers were designed by Primer Premier 5.0 software . After electrophoresis, the proteins were transferred to PVDF membrane and the membrane was incubated 37 °C with 5% skimmed milk. After incubated with a 1:2000 dilution of Rabbit anti-pGH polyclonal antibody overnight at 4 °C and incubated with AP conjugated goat polyclonal anti-Rabbit IgG (CWBIO China) at a dilution of 1:4000. Immunoreactive bands were visualized with BCIP/NBT Kit (CWBIO China) and estimated by ImageJ software. And nonspecific bands would be shielded, having no influence on quantity the target proteins.8 cells were washed in 100 mM Tris-HCl pH 8.0 and re-suspended in 300ul of yeast breaking buffer. An equal volume of acid-washed glass beads was added. Cells were disrupted by vortexing ten times for 1 min with 1 min intervals in ice. The lysate was centrifuged at 10,000 × g for 30 min at 4 °C, and supernatant was collected. Twenty microliters of the supernatant were taken for protein concentration determination using the BCA protein assay. Fifty microliters of the supernatant were then added directly to 0.95 ml of Z buffer to make a total volume of 1 mL. Then 0.2 ml of 4 mg/ml o-nitrophenyl-β-D-galactopyranoside (ONPG) stock solution was added and reactions proceeded as previous method24.Cells were harvested. A total of 1.5 × 10Supplementary data


Multicellular organisms rely on the precise and consistent regulation of gene expression to direct their development in tissue- and cell-type specific patterns. This regulatory activity involves arrays of DNA-binding transcription factors and epigenetic factors that modify chromatin structure. Among the chromatin modifiers, trithorax (trxG) and Polycomb (PcG) group proteins play important roles in orchestrating the stable activation and repression of gene expression, respectively. These proteins have generally antagonistic functions in maintaining cell and tissue homeostasis as well as in mediating widespread transcriptional reprogramming during developmental transitions. Plants utilize multiple trxG factors to regulate gene transcription as they modulate their development in response to both endogenous and environmental cues. Here, I will discuss the roles of trxG factors and their associated proteins in post-embryonic plant development. The development of multicellular organisms is driven by precise patterns of gene transcription that are tightly regulated in a spatial and temporal manner. Establishing and sustaining specific transcription states at gene loci are complex, multi-step processes. They require repertoires of sequence-specific DNA-binding transcription factors as well as epigenetic factors that alter chromatin structure and thereby affect accessibility by the transcriptional machinery. Epigenetic regulators classified as trithorax group (trxG) and Polycomb group (PcG) factors are critical for maintaining the stable transcription patterns at developmental regulatory loci by organizing chromatin in an active or inactive state, respectively . trxG anDrosophila as positive regulators of PcG developmental target genes (Table 1). The chromatin remodeling proteins include members of the SWI/SNF, ISWI, and CHD families that utilize ATP to alter nucleosome assembly and distribution  AP2 domain transcription factor (TF) genes, which are essential for root stem cell niche maintenance  maintenance. The RAM generates the entire underground root system and has a stereotypical organization. Four rarely dividing cells known as the quiescent center (QC) act as a niche  that maintenance .Figure 1A). The SET domain protein SET DOMAIN GROUP 2 (SDG2) is the major H3K4 trimethyltransferase in Arabidopsis and is necessary for genome-wide H3K4me3 deposition  and BRAHMA (BRM) also regulate RAM activity in Arabidopsis. PKL acts antagonistically to the PRC2 PcG factor CURLY LEAF (CLF) to maintain RAM stem cell activity -CLAVATA (CLV) signal transduction pathway. The homeobox TF gene WUS is expressed in the core of the meristem and confers stem cell identity on the overlying cells . In contrast, the SAND domain-containing proteins ULTRAPETALA1 (ULT1) and ULT2 restrict shoot and floral stem cell activity by limiting the size of the WUS expression domain , FLOWERING LOCUS D (FD) and SOC1, which promote the transition to flowering  and APETALA1 (AP1), which confer floral meristem (FM) identity on the primordia that form on the flanks of the primary SAM  to reproductive organs (flowers). The timing of this dynamic meristem cell fate switch is critical for plant reproductive success and occurs in response to endogenous pathways such as the age, GA and autonomous pathways (APs) as well as environmental cues including photoperiod, vernalization and temperature . The MADlowering . FT protmary SAM .FLC, SOC1 and FT are crucial to the timing of the floral transition, and a number of trxG factors are involved in these processes . Because the role of epigenetic factors in FLC regulation during vernalization has been extensively reviewed  couple removal of repressive histone marks with deposition of active marks and transcription initiation/elongation to sustain high levels of FLC expression. ATXR7/SDG25 also represses the floral transition by binding to the FLC promoter and augmenting both H3K36me3 and H3K4me3 accumulation , a core component of the Arabidopsis COMPASS-like complex (AtCOMPASS) that also binds the FLC locus and promotes its expression by elevating H3K4me3 levels   . SVP forSE (SVP) . SVP expcription . BRM repactivity . Thus thFLC transcription is crucial for the transition from the vegetative to the reproductive state. The JmjC domain-containing H3K27 demethylase REF6/JMJ12 promotes flowering independently of photoperiod by repressing FLC transcription  that produces floral meristems (FMs) instead of leaves. A small suite of floral homeotic transcription factor genes then specifies the identity of each floral organ – sepals, petals, stamens, and carpels – from the outside to the inside of the flower . The actAPETALA3 (AP3) and AGAMOUS (AG) that specify petal, stamen, and carpel identity in the developing flower , which recruit SYD to the AP3 and AG loci  by activating AP3 and AG expression, antagonizing CLF activity at the two loci by reducing H3K27me3 deposition and enhancing H3K4me3 deposition (The plant specific TF LFY and the MADS domain TF SEPALLATA3 (SEP3) play crucial roles in activating the expression of MADS box-containing floral homeotic genes such as g flower . SYD and AG loci . At the  AG loci   enhance ult1 FM phenotypes, and ALP1 promotes floral organ identity gene expression in the absence of LFY . It is becoming clear from recent research that members of all of these categories of proteins play important roles in regulating landmark post-embryonic developmental processes such as meristem maintenance and floral induction. Moreover some trxG factors, such as BRM and ATX1, mediate multiple developmental processes during plant growth whereas others appear to have more restricted roles. The coupling of a core set of reiteratively used trxG components together with stage-, tissue- or process-specific trxG components may provide a flexible mechanism for tailoring the basic process of transcription activation to discrete gene networks in response to changing endogenous and environmental signals during the life cycle.A variety of trxG factors exist in plants that carry out diverse biochemical activities to promote active gene expression states: chromatin-remodeling ATPases, histone methyltransferases, AtCOMPASS core components, histone demethylases, as well as DNA-binding and accessory proteins (Although significant progress has been made in determining the roles of trxG factors in plant development, many gaps in our understanding remain. It still remains to be determined how many trxG complexes exist in plants, not to mention their full composition and whether that composition is static or changes depending on the developmental stage or tissue. The DNA binding proteins that recruit trxG factors to developmental regulatory loci are only beginning to be identified, while elucidating the chromatin signatures of plant stem cell populations can provide a valuable starting point for determining how tissue- and stage-specific epigenetic states are ultimately achieved during development. Finally, much work remains to decipher how developmental switches between trxG and PcG activities are implemented at individual loci as well as broadly across the genome to coordinate widespread transcriptional reprogramming. Further investigation in these areas will provide a more complete picture of how plants are able to maintain and as necessary adjust their gene expression programs during development in response to a wealth of endogenous and environmental cues.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.