Drosophila brain play essential roles in regulating the proliferation of neural stem cells, neuroblasts (NBs). We found that two classes of extracellular factors, Dally-like (Dlp), a heparan sulfate proteoglycan, and Glass bottom boat (Gbb), a BMP homologue, are required for proper NB proliferation. Interestingly, Dlp expressed in perineural glia (PG), the most outer layer of the surface glia, is responsible for NB proliferation. Consistent with this finding, functional ablation of PG using a dominant-negative form of dynamin showed that PG has an instructive role in regulating NB proliferation. Gbb acts not only as an autocrine proliferation factor in NBs but also as a paracrine survival signal in the PG. We propose that bidirectional communication between NBs and glia through TGF-β signaling influences mutual development of these two cell types. We also discuss the possibility that PG and NBs communicate via direct membrane contact or transcytotic transport of membrane components. Thus, our study shows that the surface glia acts not only as a simple structural insulator but also a dynamic regulator of brain development.Despite the importance of precisely regulating stem cell division, the molecular basis for this control is still elusive. Here, we show that surface glia in the developing  Drosophila CNS develops from neural stem cells called neuroblasts (NBs)3. The self-renewal and differentiation of NBs are thought to be controlled by both intrinsic programs in the NBs as well as extrinsic cues. Despite a wealth of knowledge about intrinsic factors that regulate NB development4, very little is known regarding non-autonomous factors that affect this process. In the embryonic CNS, it has been shown that NBs receive an extrinsic signal from the overlying epithelium to properly orient the mitotic spindle axis5. Several lines of evidence suggest that post-embryonic NBs are also controlled by extrinsic factors. One previous study showed that the activin receptor, Baboon (Babo), and its transcriptional mediator, Smad2 (Smox), regulate NB proliferation in the larval CNS, indicating that activin-like ligands may play a role in this process6. Furthermore, expression of a dominant-negative form of E-cadherin in glia reduced the number of proliferating NBs7. This observation indicated that physical contact of larval NBs with glia is critical to NB proliferation. Larval glia are also known to secrete Anachronism, which maintains NBs in a mitotically inactive state (the quiescent state) during early larval stages8. Thus, although there is no direct evidence that Drosophila larval glia have a niche function, it is assumed that division and differentiation of larval brain NBs may be controlled by surrounding glial cells.The 14. HSPGs are a class of carbohydrate-modified proteins involved in a variety of biological processes such as growth factor signaling, cell adhesion, and enzymatic catalysis15. Importantly, these molecules serve as co-receptors for growth factor signaling, regulating reception and tissue distribution of secreted signaling proteins such as FGFs, BMPs, Wnts, and Hedgehog on the cell surface21. Although HSPGs are generally thought to regulate growth factor signaling on the surface of signal-receiving cells, they can also activate signaling in trans from adjacent cells to play non-autonomous roles23.Recent studies demonstrated vital roles of heparan sulfate proteoglycans (HSPGs) as important regulators of stem cell behaviorsDrosophila development and homeostasis24. BMPs are involved in numerous processes throughout all developmental stages, from patterning of the embryo26 and imaginal discs28 to stem cell maintenance29. Activin branch ligands also play diverse roles in growth and patterning in Drosophila, including control of imaginal cell proliferation32, neuroblast proliferation6, neuron remodeling34, axon guidance36, and sugar homeostasis37.The TGF-β pathway is an evolutionarily conserved signal transduction module that mediates diverse biological processes in animals, including stem cell control. Like in mammals, both the BMP and Activin branches are required for Drosophila larval brain have been classified into a few different types based on their positions, morphology, and gene expression profiles, including perineural glia (PG), subperineural glia (SPG), cortex glia, and astrocyte-like glia, which send processes into the neuropil40. Two surface glia cell types, PG and SPG, provide a structural basis for an insulation barrier between the CNS and hemolymph known as the blood-brain barrier (BBB)43. The most outer layer is formed by PG, which is characterized by the formation of extensive, thin cell protrusions. The SPG are located immediately below the PG. The SPG forms septate junctions (SJs) and are central for the Drosophila BBB function45. The BBB restricts molecular movement at the interface between blood and nervous tissues, and it plays critical roles in maintaining a regulated microenvironment for reliable neural signaling47. On the other hand, the BBB is a major obstacle for pharmacologic treatments to the brain. Past studies have focused on the functions of surface glia as an effective shield, which separates the nervous system from the open circulatory system, to maintain brain homeostasis42. However, the biological role of surface glia in regulating neural development remains to be elucidated.Glial cells in the In this study, we identified bidirectional interactions of NBs and surface glial cells in the developing larval brain. We found that Dally-like (Dlp), a HSPG of the glypican family, is expressed in PG and is critical for proper NB proliferation. Selective ablation of glial cells supported the idea that PG play an instructive role in regulating NB proliferation. We also found that Glass bottom boat (Gbb), a BMP homologue, is expressed in NBs and functions with Dlp to regulate normal proliferation of NBs. In addition to serving as an autocrine signal in NBs, Gbb also acts as a paracrine survival signal in the PG. Thus, the development of NBs and glia is controlled by reciprocal communication between these cells through TGF-β signaling pathways in the NSC niche. Finally, we discuss the possibility that PG make physical membrane contact with NBs.tout velu (ttv) and sulfateless, show a small brain phenotype mainly because the sizes of brain lobes and ventral ganglion do not properly increase during third larval instar stage. A previous study showed that mutations in activin signaling components cause a similar small brain phenotype, which is associated with altered innervation of photoreceptor (PR) axons , the Drosophila activin type I receptor, ttv mutant brains show PR axon targeting defect revealed by staining with anti-Chaoptin antibody (22B10) . We found that brains mutant for dlp, the second glypican homologue, exhibit a very severe small brain phenotype with abnormalities of PR axon projection  Fig. . This obype Fig. , or dSynion Fig. , suggestdlp mutants is caused by reduced proliferation of NBs, we analyzed NB proliferation in control and mutant brains by BrdU labeling  third instar larval brains, where NBs are actively dividing, BrdU incorporation showed a unique and robust pattern in NBs and their progeny  survive to late third larval instar stage. We found that dlp1/dlpA187 mutant brain shows significantly impaired NB proliferation  and SPG > mCD8ChRFP showed that the area of Dlp expression is largely overlapping with PG , the Drosophila dynamin48. Since dynamin is required for the formation of membrane vesicles49, expression of this dominant negative form of shi (shits) can block various types of membrane trafficking events, including endocytosis and protein secretion, and has been effectively used to disrupt glial cell function52. We expressed UAS-shits specifically in PG using PG-Gal4 (PG > shits) and assessed its effect on NB proliferation. We found that the functional ablation of PG by expression of shits severely disrupted NB cell proliferation  brains for both temperature conditions   were indistinguishable from wild-type  Fig. . Brains shits on membrane structures in PG. We observed a gross abnormality in patterns of a membrane-bound form of GFP (mCD8GFP) in PG > shits at the restrictive temperature   Fig. . Alteredshits expression. In a section near the brain surface, the PG focal plane, anti-Dlp staining showed abnormal aggregations of Dlp-containing membrane in PG > shits brain at 32 °C  as a specific marker for PG since we have found Svp expression extensively overlaps with PG-Gal4-positive cells  severely disrupted NB proliferation  mutants  show normal NB proliferation (data not shown). This synergistic effect is consistent with the notion that the gbb and dlp genes function in the same pathway to regulate NB proliferation.To determine if dad-lacZ58, a widely-used reporter of BMP signaling activity. Interestingly, we detected specific dad-lacZ signals in both Dpn-positive NBs and Svp-positive PG  can act as a trans co-receptor for Gbb (received by NB) in the brain. Since no previous work has shown a direct binding between Dlp and Gbb, we attempted to show Dlp-Gbb complex formation by co-immunoprecipitation of the proteins expressed in 59, Gbb binds to heparin-sepharose beads. Thus, similar to many other BMP members, Gbb is a heparin-binding protein59, characteristic of HS-dependent factors.We have previously shown that Dlp acts as a co-receptor for Gbb We also analyzed the cellular localization of Gbb and Dlp by expressing Gbb-HA and Dlp-Myc in dad-lacZ, Fig. 4A–Bgbb null mutant brains . We examined the genetic interactions between babo and dlp for brain growth using the PR projection assay. No synergistic effect was observed between babo and dlp mutations (data not shown), suggesting that activins regulate NB proliferation, independent of Gbb-Dlp signaling.Since both 60. Alternatively, SPG may have unknown openings, which allow PG to directly contact NBs. Although we do not know the definite answer to this question, several observations described below are consistent with these possibilities.Our results showed that PG and NBs communicate to regulate each other’s development. Specifically, our findings support the idea that Dlp serves as a co-receptor for Gbb signaling during larval brain development. However, the mechanism by which these cells communicate across SPG, the major barrier component of the BBB, is unknown. One obvious possibility is that “transcytosis” in SPG may carry specific molecules, either alone or in complexes with lipid particles, to the other side of the BBBSPG-Gal4 driver, GFP signal was detected non-uniformly, showing openings within the SPG layer  and SPG (Mdr65-LexA > mCD8GFP) often revealed regions in which the PG and SPG signals are found in the same layer  was expressed in SPG by the yer Fig. . These nyer Fig. . They doyer Fig. . Such dires Fig. . On the 61. Membrane-bound forms of spGFP proteins have been successfully used to label areas of close membrane apposition between two cells62. We developed a system to express each spGFP fragment separately in different set of cells in vivo using the Gal4/UAS63 and the LexA/LexAop64 systems. We generated and used gbb-LexA as a NB driver to express a membrane-tethered form of spGFP1-10 in NBs  system. This method is based on the fact that complementation of two split-GFP proteins, spGFP1-10 and spGFP11, reconstitutes green fluorescence while each spGFP fragment alone is not fluorescentNBs Fig. . IndepenUsing PG-driven spGFP11, we detected complementation of the two spGFP fragments, suggesting that PG cell membranes are in close proximity to NB membranes Fig. . Controlshits brain, which shows severely reduced NB proliferation  contribute to NB proliferation. The most likely candidates for these PG factor(s) are activin-like ligands, whose role in NB division has been previously reported6. We identified specific expression of Daw in PG, supporting the idea that activin signaling together with Gbb regulates NB proliferation. Altogether, glia play a “niche-like” role, contributing to the microenvironment required for normal development of contacting NBs. Glia provide Dlp to support Gbb autocrine signaling, and express activin-like ligands as additional promoters of NB proliferation. Thus, we propose a model that bidirectional communication between NBs and glia through TGF-β signaling pathways influences each other’s development in the NSC niche.Expression of p35 by Drosophila BBB60. The major Drosophila lipoprotein, lipophorin (Lpp), forms large particles with lipid molecules and transports them between different organs. In addition to lipid, Lpp particles carry sterol-linked and GPI-linked proteins. Dlp is a GPI-linked HSPG and has been shown to be incorporated into the Lpp particles70. This raises an interesting possibility that Dlp is transcytosed by the SPG through a Lpp-mediated mechanism.Our study raises a fundamental question: how can PG and NBs communicate across the BBB and affect each other’s development? Our experiments using spGFP complementation favor a model in which NBs make direct contact with PG. Thus, it is possible that Dlp acts as a trans co-receptor for Gbb at the contacting sites. However, our study does not exclude other possibilities. For example, one can expect that transcytosis in SPG would allow molecular communications across this layer. Thus, the spGFP complementation signals we observed may result from the transport of membrane components of PG and NBs. It has been shown that lipoprotein particles can cross the 71. A transcriptome study demonstrated that the surface glia express metabolic enzymes and signaling molecules at high levels in addition to various transporters, cell adhesion molecules, and ECM components72. Our study also supports the idea that the surface glia play a role not only as a simple structural insulators but a dynamic regulator of brain physiology development. Further study will be required, to fully elucidate the cellular and molecular mechanisms by which surface glia regulate the microenvironment internal to the BBB.Recent studies have shown that the surface glia are required to maintain the structural integrity of the neural lamella and to control the shape of the CNShttp://flybase.bio.indiana.edu/) except where noted. Fly strains used were: dlp1 15, dlpA187 73, gbb1 28, gbb2, gbb4, dallygem 21, ttv524 74, baboFd4 33, pros-Gal4 (a gift from F. Matsuzaki), four Gal4 drivers for glial cells: NP6293-Gal4 , NP2276-Gal4 , NP3233-Gal4, and NP2222-Gal465, daw-Gal46, gbb-Gal4, Mdr65-LexA , UAS-lacZ, UAS-dlp39.215, UAS-p35 (Bloomington Stock Center), UAS-dad58, UAS-gbb75, UAS-mCD8GFP76, UAS-ManII-eGFP77, UAS-shits 48, UAS-nlsGFP, UAS-eGFP-Dlg (a gift from V. Budnik), LexAop-rCD2::GFP64, LexAop2-mCD8GFP , vkgG454 (viking::GFP)78, gbb-lacZ, dad-lacZ58, and UAS-mCD8ChRFP. Plasmid DNAs for gbb-LexAVP16, LexAop-spGFP1-10, and UAS-spGFP11 were constructed as described below and transgenic strains bearing these constructs were generated by Rainbow Transgenic Flies, CA.Detailed information for the fly strains used is described in Flybase , rat anti-β-galactosidase , rabbit anti-β-galactosidase , mouse anti-Repo  8D12), mouse anti-Chaoptin , rat anti-Miranda (Mira) (1:100), guinea pig anti-Deadpan (Dpn) (1:1000), mouse anti-BrdU , mouse anti-Dlp , rabbit anti-active caspase 3 , mouse anti-Svp , rabbit anti-pH3, and Alexa Fluor 488, 546, 633 conjugated secondary antibodies .Larval brains were dissected in PBS and fixed with 3.7% paraformaldehyde in PBS for 30 min at room temperature. After washing with PBST (0.1% triton X-100 in PBS), tissues were blocked with 5% normal goat serum in PBST for 30 min. The samples were incubated with primary antibodies at 4 °C overnight. After washing with PBS, the samples were incubated with secondary antibodies for 2 hours at room temperature. The tissues were mounted in Vectashield (VectaLabs) and fluorescent signals were observed by confocal laser scanning microscope (Zeiss LSM710) with a 40X water immersion objective.Drosophila medium (Invitrogen) containing 40 ng/ml of BrdU (Roche) for 30 min at room temperature, and subsequently with Schneider’s Drosophila medium without BrdU for 24 hours. Tissues were washed in PBST and fixed with 3.7% paraformaldehyde in PBT for 30 min at room temperature. After tissues were treated with 1 N HCl for 30 min at room temperature, immunostaining was performed as described above. Proliferation Index (PI) was calculated as follows. The number of BrdU positive neurons from NBs in experimental brains was divided by that of wild-type control brains (yw or Oregon R). The control brains were labeled with BrdU in the same tube with experimental brain samples and the genotypes were distinguished by y+ or y− mouth hook.Larval brains were dissected in PBS and incubated with Schneider’s Drosophila gbb15 and Gbb38, and the amount of each product expressed in S2 cells is generally low. When a few cleavage sites are blocked, a larger amount of Gbb38 is produced59. There are multiple proteolytic processing sites in Gbb, which produce distinct mature ligand forms, including 79.For cellular immuno-colocalization analysis, S2 cells expressing Dlp-Myc and Gbb-HA were incubated with anti-HA and anti-Myc in a Concanavalin A-coated coverslip for 30 minutes at 25 °C, washed, and fixed with 3.7% formaldehyde. Dlp-Myc and Gbb-HA were detected as described beforeace-4p CD4-2 spGFP1-10 and rig-3p CD4-2 spGFP11, were provided by C. Bargmann62. To optimize the spGFP constructs for Drosophila, the original signal peptide (Par3) was replaced with the Drosophila myospheroid signal peptide (1-28). The spGFP11 and spGFP1-10 fragments were amplified by PCR using primers 1 and 3, and primers 2 and 3, respectively (see below), and subcloned into the pENTR/D-TOPO Gateway entry vector (Invitrogen). PCR fragments were verified by DNA sequencing. The destination vectors used for germline transformation were pTW from the Drosophila Gateway Vector Collection and pLot W from S. Diegelmann80.The original plasmids of spGFP fragments, gbb-gal4 and gbb-lacZ constructs were made by ligating gal4 and lacZ sequences downstream of the 5′ genomic sequences of the gbb gene, respectively, and subcloned into pCaSpeR3. gbb-LexAVP16 was constructed in the same manner after the LexAVP16 fragment was amplified by PCR using primers 4 and 5.Primer 1: 5′-CACCATGATCCTCGAGAGAAACCGGAGGTGCCAGCTGGCCCTCCTCATGATCGCAATACTGGCCGCCATCGCTGGACAAACGGATGCCCAGCGTGACCACATGGTCCTTCATGAGTATGTAAATGC-3′Primer 2: 5′-CACCATGATCCTCGAGAGAAACCGGAGGTGCCAGCTGGCCCTCCTCATGATCGCAATACTGGCCGCCATCGCTGGACAAACGGATGCCCAGATGTCCAAAGGAGAAGAACTGTTTACC-3′Primer 3: 5′-CTAGCGCCTTCGGTGCCGG-3′Primer 4: 5′-GTGGCGGCCGCTCCGTCCAAAACACGAAATAATGAAAGCGTTAACGGCCAGGCAACAAGAG-3′Primer 5: 5′-GTGGCGGCCGCCTACCCACCGTACTCGTCAATTCCAAGGGC-3′All data generated or analyzed during this study are included in this published article and its Supplementary Information files.Supplemental Information


Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300–400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression. Actin is the most important structural protein in the cell with multiple functions . There aDNA into structural units called nucleosomes. Histones are the main protein components of chromatin, spool around DNA, and play a role in gene regulation. There are five major families of histones: H1/H5, H2A, H2B, H3 and H4. H2A, H2B, H3 and H4 are the core, while H1 and H5 are linker histones. All core histones contain the histone fold domain containing three alpha helices linked by two loops ,  and andDNA iwo loops . Howeverwo loops . Histonewo loops , 31] an anDNA inwo loops . Histonewo loops  and H2A-wo loops . H2A hiswo loops . H1 histwo loops , may indSGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYSERVGAGAPVYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGRVTIAQGGVLPNIQAVLLPKKTESHHKAKGK), molecular mass (14000 Da) and structure to investigate histone-actin interactions. We showed that both histone preparations polymerize G-actin, bundle F-actin and significantly increase its viscosity at substoichiometric or stoichiometric concentrations. We found that several factors, including ionic strength, cofilin, DNase1 and DNA affect the structure and stability of the histone induced F-actin bundles. The results indicate that histones are tightly bound to F-actin by strong electrostatic and hydrophobic interactions and affect actin structure.In this study, we used a histone mixture preparation, which represents histones secreted from the cells during apoptosis. We used also H2A histone with known molecular characteristics including sequence (N-(1-pyrene) maleimide, Alexa488 SE were obtained from Molecular Probes . DNase1, ATP, ADP, dithiotreitol (DTT), histone type III (histone mixture) and deoxyribonucleic acid (DNA) from calf thymus were purchased from Sigma Chemical Co. . Acetone dry powder was purchased from Pel-freeze Biologicals . Human recombinant H2A histone was bought from New England Bio Labs . Viscous Aqua was purchased from Ursa BioScience . Yeast cofilin was a generous gift of Prof. Emil Reisler, 2, 0.2 mM ATP, 0.5 mM β-mercaptoethanol, pH 8.0 (CaATP-G-buffer). MgF-actin was polymerized from CaATP-G-actin by incubation with 2 mM MgCl2 at room temperature for 30 min. MgF-actin was diluted for further treatments in MgF-buffer containing 5 mM MOPS, 2 mM MgCl2, 0.2 mM ATP and 0.5 mM DTT, pH 7.4. The concentration of unlabeled rabbit skeletal muscle CaATP-G-actin and Mg-F-actin was determined spectrophotometrically using the extinction coefficients E1%290nm = 11.5 cm-1. . Molecular masses of skeletal actin, yeast cofilin and H2A histone were assumed to be 42 kDa, 15.9 kDa and 14.1 kDa, respectively.CaATP-G-actin was prepared from acetone dried powder derived from the back and leg muscles of rabbit by the method of Spudich and Watt  that yieɛ344 nm = 22000 cm-1M-1 was, 100%.Labeling of Mg-F-actin at Cys-374 with pyrene maleimide was carried out according to Kouyama and Mihashi  with som2, 0.2 mM ATP) for 3 h to remove DTT and to adjust the pH. Filaments were incubated in labeling buffer with a 3-7-fold molar excess of dye overnight at 4°C. Labeled F-actin was pelleted in TLA110 rotor, 60,000 rpm, for 30 min then resuspended in CaATP-G-buffer, and dialyzed for 48 h versus the same buffer. Labeled actin monomers were centrifuged in TLA110 rotor, 60,000 rpm, 30 min followed by gel-filtration (Superdex S200 10/300 GL). The label concentration was calculated using ɛ495nm = 71,000 M−1cm−1.Labeling was carried out according to Mahaffy et al.  and GrinActin polymerization was followed as increase in fluorescence of pyrene-labeled G-actin , which wFor monitoring extent of polymerization samples were centrifuged at 129,151xg for two hours (high speed centrifugation) after addition of histone mixture or H2A histone to actin. For monitoring the extent of F-actin bundling samples were centrifuged at 20,800xg for 8 min (low speed centrifugation). The supernatants were run on SDS-PAGE and analyzed by densitometry with TINA 2.07d software.2). Actin polymerization was started by addition of polymerizing salts and proteins (or corresponding buffers) to Mg-ATP-G-actin; the resulting mixtures (3 CV) were introduced into the flow chambers. After 15–30 min of on-slide polymerization (at room temperature) images of the random fields were taken. All TIRF data was analyzed using ImageJ (Fiji) software . Background subtraction was done using rolling ball radius algorithm (50 pxls). The images were converted into black-and-white 8-bit format then the colors of the objects and background were inverted for clarity of presentation.Untethered 15% Alexa488 SE labeled actin filaments were imaged on Pluronic F127-coated surface . Single Student’s t test and one-way ANOVA were used for calculation of p values in evaluation of significance. * = p<0.05, ** = p<0.01, *** = p<0.005. Unless specified, all presented data are mean ± SD of three independent experiments performed in triplicate. All presented SDS gels blots are representative of three independent experiments.Monovalent, divalent and polyvalent cations are well known to polymerize G-actin to actin filaments. The polymerizing activity of several positively charged polycationic antimicrobial polypeptides, including lysozyme and LL-37 were studied , 11]. W. W11]. WF-actin was shown to form bundles upon addition of polyvalent cations  because Polycation induced F-actin bundles are sensitive to ionic strength and unbundle (dissociate) with increase in the concentration of monovalent cations  because DNase1 is a G-actin binding protein. It binds very tightly to the D-loop of G-actin and forms co-crystals with actin monomers  and depoStaphylococcus aureus nuclease, was added to histone mixture or histone 2A bundled F-actin  H2A histone for 15 minutes  H2A histone and 1 μM cofilin for 15 minutes  and/or to additional hydrophobic interactions. Lower concentration of histones are needed for actin bundling than of other polycations as, LL-37, lysozyme , polylysThe mechanism of action of agents affecting the stability and structure of F-actin–histone bundles was studied. These agents affect the bundles by different mechanisms. NaCl increases ionic strength and dissolves the bundles by masking electrostatic interactions between histones and actin filaments , 44]. D. D44]. DTwo histone preparations were used in the present experiments. Histone mixture, which represents histone released from dead cells following apoptosis and NET formation , and recThe high stability and viscosity of the histones induced actin bundles may have pathophysiological significance in cystic fibrosis, which is the most common fatal inherited disease in the western world . Actin bThe actin histone interaction might have significance also in cell physiology. The binding of histones to nuclear actin could affect gene activation. It was reported that monomer nuclear actin inhibits while polymer nuclear actin increases the activity of class 1 histone deacetylase . Histone


Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly. Accurate segregation of chromosomes from a mother cell to its two daughters during cell division is a prerequisite for healthy cell physiology and for the transmission of the genetic information across generations . SpecialKnl1, Mis12, and Ndc80 complexes , which replaces histone H3 in nucleosomes . The priomplexes . The cenomplexes   with histones H2A, H2B, and H4. In vitro, these interact specifically and selectively with two CCAN components, CENP-C and CENP-N . BindingCENP-N forms a constitutive complex with CENP-L (designated CENP-LN complex), which in turn interacts with the CENP-HIKM complex and with CENP-C . BindingNCP (1-212) also bound selectively to CENP-ANCPs but not H3NCPs   was sufficient to interact with CENP-L   to deducID 4JE3)  and a C-ID 4JE3) . Iml3 heID 4JE3)  (HinshawID 4JE3) . Due to 4JE3) . Structural similarities of the CLN-HD suggest that CENP-N and CENP-L are evolutionary related. However, sequence identity of the two domains, even after structural superposition, is minimal, likely explaining why structural similarity had not been predicted  , we obtained a three-dimensional reconstruction of CENP-Nt ~4.0 Å . We builID 3AN2) , combineID 3AN2) , and thecleosome .NCP (PDB ID 3AN2)  may be flexible. Of the ~2400 Å2 of CENP-ANCP and CENP-N surface area that become buried in the complex,~1400 Å2 are at the CENP-N:DNA interface, where both CENP-N domains form extensive interactions with DNA from bp −21 to −35 relative to the twofold axis, or superhelical location [SHL] −2 to −3. There is a marked accumulation of positively charged residues on this DNA binding interface   . CENP-N,ID 3AN2) . There interface . Four lonterface . The hignterface   . Likely,g assays , reflectNCPs from H3NCPs, because CENP-N bound selectively to CENP-ANCPs even when the CENP-ANCPs and H3NCPs contained the same DNA sequence  mutant of R11 , including R11A and two double mutants (E3A-E7A and K143A-Y147A), lost the ability of CENP-NCENP-Nwt . CollectNCPs cooperatively, that is with increased binding affinity in comparison to any of the individual subunits or sub-complexes  for its potential role in CENP-N binding. A 5-alanine mutant of residues 302–306 (identified as CENP-C5A) failed to interact with CENP-NL, identifying this region of CENP-C as the CENP-LN binding motif  of 1 µM, but it showed no binding to CENP-C225-364-5A  or mutant (5A) copies. Depletion of CENP-C prevented kinetochore localization of CENP-N, showing that nucleosome binding is not sufficient for CENP-N to reach kinetochores at the low cellular concentration of these proteins. Exogenously expressed wild-type CENP-C promoted CENP-N recruitment, while CENP-Comote it . Thus, tNCPs are the only known direct and specific points of contact of the kinetochore with the centromere and are therefore the crucial effectors through which CENP-A implements its role   , the CEN 4 × 23) . Thus, C 4 × 23) .NCP. It has also been proposed that CENP-C reshapes and rigidifies the CENP-A nucleosome and that it modulates the DNA termini to make them match the loose wrap observed at centromeres . At present, we cannot definitively conclude whether the stabilization of the termini is due to CENP-N binding to the CENP-A nucleosome, as we have not yet been able to obtain a high-resolution EM structure of the CENP-A nucleosome in isolation for comparison. It is possible that the cryogenic conditions used for our structural work stabilize a specific conformation of the complex.It has been proposed that CENP-N is significantly stabilized upon binding to the CENP-A nucleosome . Our stutromeres . Importatromeres ]. We alsIn most organisms, centromere identity is not specified by the centromere’s DNA sequence, but rather by the enrichment of CENP-A at a defined chromatin domain. De novo formation of stably inherited centromeres at previously non-centromeric sites (neo-centromeres) provides clear evidence in favor of this idea. Thus, rather than being genetically (i.e. DNA-sequence) specified, centromeres are epigenetically specified, with the pre-existing enrichment of CENP-A being a necessary condition for continued deposition of new CENP-A at the same site through the generations. There is therefore considerable interest in the molecular mechanisms that promote new CENP-A deposition at centromeres during the cell cycle, and in the mechanisms that promote the stabilization and persistence of CENP-A after its incorporation at centromeres.S. cerevisiae) and an adaptor complex consisting of the Mis18 and M18BP1 subunits, has been described in recent years  at 4°C for 2 hr. After extensive washing with lysis buffer, the GST fusion proteins were eluted in lysis buffer supplemented with 20 mM reduced glutathione. The eluted proteins were concentrated in a 10 K Da Amicon-Ultra-15 Centrifugal filter  either alone or in the presence of GST-tagged 3C protease. Concentrated proteins were subsequently loaded onto a Superdex 200 16/600 column equilibrated in 20 mM HEPES pH 7.5, 2.5% glycerol, 300 mM NaCl and 1 mM TCEP. The corresponding peak fractions were collected and concentrated in a 10 KDa MWCO concentrator and then flash frozen in liquid N2, and stored at −80°C until further use.GST-CENP-L and its complexes with CENP-N fragments were produced in insect cells. Specifically, the coding sequence expressing 3C cleavable GST-tagged CENP-L was sub-cloned into MCS2 of the MultiBac vector pFL , while tisolated . BrieflyNT constructs  were generated by introducing a sequence encoding 6His followed by two stop codons at the designated positions. CENP-NNT constructs were expressed in SF21 insect cells . Pellets from 1 L of cell culture (~2 million cells/ml) were lysed in 100 ml lysis buffer . Nickel NTA beads (1.5 ml) were incubated with the lysed cells overnight at 4°C. Beads were washed with wash buffer , and eluted with elution buffer . The concentrated eluate was loaded onto an S200 gel filtration column in buffer containing 20 mM Tris.Cl pH 7.5, 300 mM NaCl, 10% glycerol and 1 mM DTT for the final purification.Full length CENP-N was cloned into a pACEbac1 vector. CENP-N1-212. Mutant CENP-N1-212 constructs were created by site-directed mutagenesis using the QuikChange kit . Escherichia coli (DE3) cells harboring vectors expressing CENP-N1-212-His or CENP-N1-235-His were grown in TB media supplemented with 100 µg ampicillin at 37°C at an OD600 of 0.8–1.0. Then the temperature was reduced to 20°C and protein expression was induced with 0.3 mM IPTG for 16 hr. Cells were harvested at 4600 g for 15 min. Bacterial pellets were resuspended in lysis buffer containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, 10 mM MgCl2 and 5 mM imidazole. Resuspended cells were lysed by sonication and cleared by centrifugation at 100,000 g at 4°C for 30 min. The cleared lysate was incubated with cOmpleteTM his tag beads (Sigma-Aldrich) and incubated at 4°C for 2 hr. After extensive washing, CENP-N1-212-His or CENP-N1-235-His were eluted in lysis buffer supplemented with 300 mM Imidazole. A 6 ml ResourceS cation exchange column was pre-equilibrated in 15% buffer B  and 85% buffer A . CENP-N1-212 -His or CENP-N1-235-His were diluted with buffer A to reach a final concentration of 150 mM NaCl, loaded onto the ResourceS column .Fragments encoding different constructs of CENP-N were sub-cloned from a cDNA into pST50Tr-DHFRHIS for expression of recombinant C-terminally polyhistidine-tagged products . To prod2-545 or CENP-C225-364 were obtained from codon-optimized CENP-C cDNA and subcloned in pGEX-6P-2rbs for 3′ fusion to the sequence encoding GST. Mutant CENP-C constructs were created by site-directed mutagenesis using the QuikChange kit . Expression and purification for all CENP-C constructs and mutants was carried out as described  wrapped around CENP-A or H3 octamers was a gift from C.A. Davey. Purification of CENP-A or H3 containing NCPs were performed as previously described  were incubated with either NCPs or CENP-C as prey at a 3 μM concentration. The bait was loaded onto 10 µl pre-blocked beads before adding the prey. The reaction volume was topped up to 40 μl with buffer and incubated at 4°C for 1 hr. Beads were spun down at 500 g for 3 min. The supernatant was removed and beads were washed twice with 200 μl pull-down buffer supplemented with 0.05% Triton-X-100. Supernatant was removed completely, samples boiled in 15 µl Laemmli sample loading buffer and run on a 14% SDS–PAGE gel. Bands were visualized with Coomassie brilliant blue staining. For pre-blocking of GSH sepharose beads, 750 μl of GSH sepharose beads were washed twice with 1 ml washing buffer  and incubated in 1 ml blocking buffer  overnight at 4°C with rotation. Beads were washed five times with 1 ml washing buffer and resuspended in 500 μl washing buffer to have a 50/50 slurry of beads and buffer. Pull-down assays were repeated at least three times.GST pulldown assays were performed as previously described  with minAnalytical SEC was performed on either Superdex 200 5/150 or Superose 6 5/150 columns in a buffer containing 20 mM HEPES (pH 7.5), 300 mM NaCl, 2.5% glycerol, 2 mM TCEP, pH 7.5 on an ÄKTAmicro system . Proteins were mixed at 6 μM in a total volume of 50 μl, incubated for one hour on ice, spun for 15 min in a bench-top centrifuge before each injection. All samples were eluted under isocratic conditions at 4°C in SEC buffer  at a flow rate of 0.1 ml/min. Elution of proteins was monitored at 280 nm. Fractions (100 μl) were collected and analyzed by SDS–PAGE and Coomassie blue staining.NT (CENP-N:CENP-A nucleosome complex) were generated by mixing CENP-A nucleosome and CENP-NNT at a molar ratio of 1:3, then dialyzed against 20 mM Tris.Cl pH 7.5, 50 mM NaCl, 1 mM EDTA and 1 mM DTT. The complexes were concentrated to approximately 2.5 µM using a Millipore concentrator (MW 50 kD cut-off).Complexes containing CENP-A nucleosome and CENP-NNT constructs in buffer containing 20 mM Tris.Cl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM DTT and 0.1% CHAPS for 30 min at room temperature. The mixture was analyzed by 5% native PAGE, and the gel was stained with SYBR Gold. The gel was imaged at 488 nm (for SYBR Gold staining or Alexa488 labelled H2B in the nucleosome) and 647 nm (for Atto N 647 labeled H2B in the nucleosome) using a Typhoon imager . Intensity at 647 nm was used for binding curves, with the number of replicates indicated. The intensity of the nucleosome band (not the shifted bands) was quantified, and normalized to the nucleosome sample without CENP-N.Nucleosomes were mixed with CENP-NAll protein samples were loaded onto a Superdex 200 10/300 column equilibrated in 20 mM HEPES pH 7.5, 2.5% glycerol, 300 mM NaCl and 1 mM TCEP prior to ITC runs. ITC measurements were performed at 25°C on an ITC200 micro calorimeter . In each titration, the protein in the cell (at a 5–8 µM concentration) was titrated with 19 × 2 µl injections  of protein ligand (at 50–80 µM concentration). The injections were continued beyond saturation to allow for determination of heats of ligand dilution. Data were fitted by least-square procedure to a single site binding model using ORIGIN software package .36 the cDNA sequence was ligated with the PCR amplified pCDNA5/FRT/TO-EGFP-IRES or pCDNA5/FRT/TO-mCherry-IRES to create the final constructs used in this study. Each plasmid was then sequence verified before use.Plasmids for stable cell lines were generated in pCDNA5/FRT/TO-EGFP-IRES, a modified version of the pCDNA5/FRT/TO vector . The starting plasmid for EGFP expression was made by PCR amplifying the EGFP sequence of the pEGFP-C1 plasmid  and cloning it into the pcDNA5/FRT/TO vector previously modified to carry an internal ribosomal entry site (IRES) sequence creating the pcDNA5/FRT/TO EGFP-IRES vector . Mammaliwt or mutants with a C-terminal-mCherry tag transfected into asynchronously growing cells and expressed for 24 hr before preparation for immunofluorescence analysis. To analyze whether the CENP-C5A mutant abrogates kinetochore localization of CENP-N, we used Flp-In T-REx HeLa cells  to generate stable doxycycline-inducible cell lines, which were maintained in DMEM  with 10% tetracycline-free FBS (Clontech) supplemented with 50 μg/ml Zeocin (Invitrogen) and 2 mM L-glutamine (PAN Biotech). Flp-In T-REx HeLa expression cell lines were generated as previously described (WT or GFP-CENP-C5A mutant and CENP-C siRNA for 72 hr. When required 16 hr before fixation cells were synchronized overnight in 0.33 µM nocodazole (Sigma–Aldrich) to assess mitotic localization. Endogenous CENP-C was depleted by siRNA (target sequence: 5′-GGAUCAUCUCAGAAUAGAA-3′ obtained from ThermoFisher), transfected into cells using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher) as per the manufactures instructions for 72 hr.U2OS cells, a gift from A. Bird , were grown in DMEM  supplemented with 10% FBS , penicillin and streptomycin , and 2 mM L-glutamine (PAN Biotech). Transient transfections were performed using pCDNA5-FRT-TO plasmids carrying either CENP-Nescribed . FlpIn TEither U2OS or FlpIn T-REx HeLa cells were fixed using 4% paraformaldehyde in phosphate buffered saline (PBS), permeablised using 0.25% Triton X-100 in PBS and blocked in 3% BSA/PBS. U2OS cells were stained for with the following antibodies: anti-CENP-C [rabbit polyclonal antibody SI410, diluted 1:1000 ; and CRE1-212His6  or CENP-N1-235His6 were obtained in sitting drop crystallization experiments at c.a. 6 mg/ml in a 96 well format using a Mosquito protein crystallization robot (TTP Labtech) at 4°C. Crystals grew in various commercial screens including Qiagen Nextal PEGSII conditions B11 and B12 and Qiagen Nextal PEG conditions H6 and H8 within 24–48 hr, reaching maximum size in 5–7 days. CENP-N1-235His6 crystals were further optimized in 24-well plates via hanging drop method using a two-dimensional grid screen varying PEG3350 (from 6–16%) and pH (from 6.6 to 7.2). Selenomethionine (SeMet) derivatives of CENP-N1-235His6 were crystallized under similar conditions. Crystals were cryo-cooled in a mother liquor solution containing 20–25% (v/v) glycerol. All data were collected at 100K using a Pilatus 6M detector either at the X10SA beamline at the SLS in Villigen, Switzerland, or at the P11 beamline of PETRA in Hamburg, Germany. All data sets were integrated and scaled using XDS and XSCALE  crystals grew in space group P4h PHENIX  located h PHENIX  successfh PHENIX  and PHENMonomers A and B in the asymmetric unit are quite similar except for a minor hinge-bending between the two subdomains of each monomer, and except for loops 137–142 and 164–174. In monomer B, the 137–142 loop is pulled approximately 3 Å away from the remainder of the molecule by a symmetry contact, and in turn pulls on the neighbouring loop causing a 2.6 Å movement of the backbone of K117. This loop has no crystal contacts in monomer A. Similarly, residues 164–174 of monomer B pack against a symmetry related molecule, most likely causing the observed (relative) stabilization of M167 in monomer B and backbone shifts of up to 7.3 Å relative to monomer A. Residues 166–168 have very weak density especially in chain A, corresponding to a weak and multiple anomalous density of SeM167, indicating multiple conformations of this loop. The coordinates of CENP-N have been submitted to the protein data bank (PDB) with code 6EQT.NT:CENP-ANCP complex (2.5 µM concentration). The grids were glow discharged  at 40 mA for 20 s. 4 µl sample was applied onto the grid before plunge freezing into Ethane, using a Vitrobot . Blot time was 4 s. All grids were stored in liquid nitrogen before imaging. CENP-NNT:CENP-ANCP complex was imaged at nominal magnification of 29000x on a FEI Titan Krios (300 kV), equipped with a Gatan K2 Summit direct detector. Pixel size was 1.02 Å. The movies were captured in super resolution mode with electron dose rate at 10 electrons per pixel per second for 8 s and 0.2 s per frame. Defocus range was −1.0 to −2.5 µm.Quantifoil 2/2 grids  were used for the CENP-NMotioncor2 was used for the alignment of images (motion correction) . GCTF waThe original 3D refined map and post-processed map were used for the model fitting and refinement. DNA was taken from a high-resolution crystal structure of a nucleosome with 601 DNA [Protein Data Bank accession code 3LZ0 , CENP-A  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.eLife. Your article has been reviewed by three peer reviewers, one of whom, Geeta J Narlikar (Reviewer #1), is a member of our Board of Reviewing Editors, and the evaluation has been overseen by John Kuriyan as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Hongtao Yu (Reviewer #2).Thank you for submitting your article "Decoding the centromeric nucleosome through CENP-N" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see the reviewers agree that your work is a significant advance in understanding the structural basis for how kinetochores are assembled on centromeres. They have some comments that we would like you to address. We anticipate that you can address these comments with either textual clarifications or re-analysis of some of the existing data.SummaryFunctional centromeres are epigenetically marked by the histone H3 variant CENP-A and form the foundation for the assembly of the kinetochore, which provides landing pads for spindle microtubules and spindle checkpoint proteins. Proper assembly of the kinetochore is thus critical for accurate chromosome segregation and genomic stability. The constitutive centromere associated network of proteins (CCAN) directly recognizes CENP-A-containing nucleosomes and builds the inner kinetochore. Two CCAN components, CENP-C and CENP-N, have been shown to specifically and cooperatively bind CENP-A nucleosomes. The structure of the CENP-A nucleosome and of a CENP-C fragment bound to the CENP-A nucleosome has been reported previously. CENP-N was the first CCAN protein identified that selectively binds CENP-A nucleosomes in chromatin. How CENP-N achieved this selective binding has been a mystery until this work. Further how CENP-N and CENP-C cooperate to specifically recognize CENP-A nucleosomes was also not known.This manuscript makes 3 major contributions: 1) It provides the X-ray crystallographic structure of the CENP-A nucleosome binding region of CENP-N; this allowed the authors to assign two structural domains (the PYRIN domain and the CLN-HD) which could not be predicted from sequence alone. 2) It provides the first insight into how CENP-N binds to CENP-A nucleosomes by solving a Cryo-EM structure of CENP-N bound to the CENP-A nucleosome. While there was indication that the L1 loop region of CENP-A was involved in the interaction, this work shows how CENP-N engages the nucleosome and also discovers a surprisingly large DNA-interacting domain. This binding mode suggest that CENP-N can antagonize the nucleosome destabilizing action of chromatin remodelers. 3) This work furthers our understanding of the interplay between CCAN proteins, in particular interaction between the CENP-L/N complex and CENP-C through a conserved CENP-C motif, and how their interactions contribute to centromere assembly.This work provides a structural explanation of why some previously reported mutations in CENP-N  break the interaction of CENP-N with the CENP-A nucleosome and fail to target CENP-N to centromeres. The structural studies are supported by in vitro biochemistry to probe the interactions and cell biology experiments to test their relevance in vivo. This work also brings up many interesting questions that are beyond the scope of this manuscript. With the identification of the PYRIN domain, does CENP-N interact with itself or other PYRIN domain containing proteins since PYRIN domains have been found to be protein-protein interaction domains? Is CENP-N binding affecting the conformation of DNA ends in CENP-A NCPs and if so, how? With the ability to potentially separate the functions of CENP-C , how do these different 'activities' contribute to overall centromere assembly and function? Overall the findings and new questions that are raised greatly advance the molecular understanding of centromere and kinetochore assembly.Essential revisions:The authors perform mutational analysis to functionally validate their structural information. In general, the trends support the structural insights since mutating a few of the interacting residues caused at least some defect in either pull down assays or localization assays. However, the following points should be further clarified.1) A naïve assumption is that CENP-N localization to centromeres would depend on its ability to selectively recognize CENP-ANCPs over H3NCPs. However, while the authors see that a CENP-N E3A/E7A mutant weakens CENP-N's affinity for CENP-ANCPs, oddly more so than its affinity for H3NCPs, they do not examine the localization of this mutant. This data is important to confirm the validity of nature of the CENP-N – CENP-ANCPs specific interaction suggested by the structures. Can the authors clarify why they did not look at localization of the CENP-N E3A/E7A mutant? In addition, quantification and statistical testing of In 2) There is some concern that normalizing to CREST staining in 3) Related to point 2, showing that expression levels of the different mutants are similar would allay the concern that the localization differences are simply due to expression level differences.4) The largest decrease in fraction bound in  Essential revisions:The authors perform mutational analysis to functionally validate their structural information. In general, the trends support the structural insights since mutating a few of the interacting residues caused at least some defect in either pull down assays or localization assays. However, the following points should be further clarified.1) A naïve assumption is that CENP-N localization to centromeres would depend on its ability to selectively recognize CENP-ANCPs over H3NCPs. However, while the authors see that a CENP-N E3A/E7A mutant weakens CENP-N's affinity for CENP-ANCPs, oddly more so than its affinity for H3NCPs, they do not examine the localization of this mutant. This data is important to confirm the validity of nature of the CENP-N – CENP-ANCPs specific interaction suggested by the structures. Can the authors clarify why they did not look at localization of the CENP-N E3A/E7A mutant? In addition, quantification and statistical testing of We agree with the reviewers that this point needed clarification. We have now added new EMSA experiments in a new NCPs but not for H3NCPs, like all other CENP-N mutants at the interface with CENP-A. This is expected: CENP-N has an extensive interface with DNA, and in the EMSA assays at low salt this triggers measurable binding also to H3NCPs. We show that this “background” binding to H3NCPs, contrarily to binding to CENP-ANCPs, is not affected by mutations targeting the CENP-A binding interface of CENP-N. This explains why these mutations  affect CENP-ANCP binding more than they affect H3NCPs binding, and we now clarify this in the text.In the new We did not include the E3A-E7A mutant in our collection of mutants studied in vivobecause the time constraints imposed by the competing paper made it difficult to harmonize the diverse sets of mutants that were generated in the two collaborating labs. We believe that demonstrating the effects of all mutants in all assays will not contribute to the conclusions of our manuscript, as these experiments collectively, and conclusively, validate the nucleosome-CenpN interfaces determined by cryo-EM.In We apologize for the confusion. The pull down data for the Arg11 mutant was already shown in 2) There is some concern that normalizing to CREST staining in We agree that this might have been a concern, had we been working under conditions of RNAi of endogenous CENP-N. However, the experiment was carried out in presence of endogenous CENP-N, i.e. we were only measuring the relative ability of mCherry-labeled wild type and mCherry-labeled mutant constructs to reach the kinetochore in presence of endogenous CENP-N. Under these conditions, we do not expect any loss of CENP-C or other antigens recognized by the CREST serum, as endogenous CENP-N is available to “supplement” a requirement for this protein even if the mutants cannot reach the kinetochore.3) Related to point 2, showing that expression levels of the different mutants are similar would allay the concern that the localization differences are simply due to expression level differences.Because of time constraints, we were not able to include a western blot to demonstrate with a single gel that the expression levels of mutants tested in the localization studies in cells are similar, . However, we are confident that the expression levels are similar based on individual mutant plasmid titration experiments. We now write that the expression of mutants was similar and refer to these data as “unpublished data”4) The largest decrease in fraction bound in K143 was selected because it is positioned at the interface with the CENP-A L1 loop. We now indicate this more clearly in the text. We agree with the reviewers that the choice of mutants is less logical than one might have hoped, which, as explained above, was in part due to the fact that different sets of mutants were generated independently in the two collaborating labs. We note however that the situation is not as bad as it might seem. However, to reconcile our datasets, we have now created a “standard” with the R11A mutant, which is used across all assays. Second, we show through the EMSA assay that all mutants that affect the direct interaction of CENP-N with the CENP-A L1 loop cause a decrease in binding selectivity for CENP-A, as expected. Finally, we show that mutations at the interface with the L1 loop, alone or in combination with mutation in the DNA binding region of CENP-N, cause displacement from the kinetochore. We believe that overall this offers a clear picture of the effects of mutations on various functional interfaces of CENP-N, and present a strong validation for the identified interfaces.


Expression of HLA-C varies widely across individuals in an allele-specific manner. This variation in expression can influence efficacy of the immune response, as shown for infectious and autoimmune diseases. MicroRNA binding partially influences differential HLA-C expression, but the additional contributing factors have remained undetermined. Here we use functional and structural analyses to demonstrate that HLA-C expression is modulated not just at the RNA level, but also at the protein level. Specifically, we show that variation in exons 2 and 3, which encode the α1/α2 domains, drives differential expression of HLA-C allomorphs at the cell surface by influencing the structure of the peptide-binding cleft and the diversity of peptides bound by the HLA-C molecules. Together with a phylogenetic analysis, these results highlight the diversity and long-term balancing selection of regulatory factors that modulate HLA-C expression. HLA-C expression levels correlate with immune responses to pathogens and autoimmunity, and vary in an allele-specific manner across individuals. Here the authors identify factors that drive differential expression of HLA-C allomorphs at the cell surface, and influence the structure of the peptide-binding cleft and diversity of peptides bound by HLA-C molecules. HLA) gene locus is one of the most diverse regions of the human genome, with extreme polymorphism and associations with a large number of human diseases24The human leukocyte antigen  expressed on natural killer (NK) cells2789HLA class I molecules, encoded by HLA-C expression varies widely in an allele-specific manner4412134HLA-C (-35 C/T) was correlated with HIV-1 viral load and HLA-C expression in people of European ancestry1214HLA-C, which is a polymorphic microRNA binding site for miR-148a. HLA-C alleles that have an intact miR-148a binding site, such as C*07 and C*03 among others, have low expression as a result of inhibition by the microRNA, whereas other HLA-C alleles  that escape miR-148a binding due to a deletion in the miR binding site, are expressed at higher levels. This insertion/deletion polymorphism in the 3′UTR of HLA-C is only fractionally responsible for the differential surface expression of HLA-C allelesHLA-C alleles with an intact miR-148a binding site, and has no impact on those alleles that escape miR-148a regulationHLA-C show a continuous rather than a bimodal expression pattern, suggesting that additional factors with stronger effects than the miR binding site are primarily responsible for influencing differential HLA-C surface expressionThere has been a wide interest in identifying factors that influence differential expression of HLA-C molecules. A single nucleotide polymorphism 35 kb upstream of HLA-C alleles, C*05 and C*07, which are commonly found at allele frequencies ranging between 3–14% (C*05) and 18–38% (C*07) in Caucasian populationsHLA-C allomorphs. This regulation is found to be post-transcriptional as the differential cell surface expression does not correlate with mRNA levels. Furthermore, we observe that HLA-C*07 has a deeper and narrower antigen-binding cleft than the relatively flat peptide-binding cleft of HLA-C*05. In line with this, HLA-C*05 binds a larger range of peptides than HLA-C*07, which can stabilize it on the cell surface, hence offering a potential explanation for the differential cell surface expression of these HLA-C allomorphs.To further understand the mechanisms responsible for differential HLA-C expression, we chose two HLA-C molecules, we selected two common HLA-C alleles, HLA-C*05:01:01:01 (referred to as C*05) and HLA-C*07:02:01:03 (referred to as C*07), that differ in expression levels, and have either a disrupted (C*05) or intact (C*07) miR-148a binding site, respectively. To study the involvement of the different parts of the HLA-C gene in contributing towards differential surface expression, we generated hybrid C*05 and C*07 genomic constructs. One half of these hybrid constructs, consisting of the promoter, 5′UTR, exons 1–3 and introns 1, 2 and part of intron 3, was taken from the HLA-C*05 or HLA-C*07 alleles, while the second half of the constructs were identical, and consisted of part of intron 3, exons and introns 4–8, and the 3′UTR of the murine H-2Kb allele  upstream of the luciferase gene in a promoter-less vector (C*07 led to a significantly (∼2-fold) higher expression of the luciferase reporter gene in comparison to the C*05 promoter  that we used to stain for HLA-C expression has monomorphic specificity and binds fully assembled HLA class I molecules with equal affinity21C*05 and C*07 constructs including the sequence of exons 1–8 of these alleles were generated Within the HLA-C*05-SAE complex, there are four main anchor residues, P1-Ser, P3-Glu, P7-Leu and P9-Leu. The P1-Ser is surrounded by seven aromatic residues, arising from the floor of the antigen-binding cleft  and from the α1 and α2 helices , as well as hydrogen bonding to Lys66 . The P3-The structure of HLA-C*07 in complex with the RYR peptide revealed canonical P2-Tyr and P9-Leu anchor residues, a large network of interactions at P1-Arg and a secondary anchor residue at P3-Arg. The P1-Arg in HLA-C*07 was stabilized by aromatic residues, similarly to the P1-Ser of the SAE peptide in HLA-C*05, with an additional salt bridge formed with the Glu63 . The larHLA-C*05 and HLA-C*07 differ by 22 residues, of which 15 are located within the α1/α2 domains, with ten of these being involved in peptide interactions, namely Tyr9, Thr73, Asn77, Lys80, Tyr99, Asn114, Phe116, Trp147, Glu152 and Arg156 . While H3, 3, Overall the B and F pockets, binding the characteristic anchor residues at P2 and the C-terminus of HLA class I-restricted epitopes, contain large aromatic residues in HLA-C*05 that are absent in HLA-C*07. Consequently, the antigen-binding cleft of HLA-C*05 is composed of residues with large side chains, and accordingly offers a more shallow cleft C*05 and C*07 transfected 721.221 cells, and sequenced bound peptides by mass spectrometry.To compare the peptide repertoire of HLA-C*05:01 and HLA-C*07:02, we isolated these HLA class I molecules from the cell surface of equal numbers of A total of 1,870 specific peptides were identified from HLA-C*05 molecules . The majA total of 580 specific peptides were identified from HLA-C*07 molecules . The majIn line with the structural analysis, these peptide-repertoire data demonstrate that the number of distinct peptides bound by HLA-C*05 were threefold higher than those bound by HLA-C*07, in agreement with the higher relative expression of HLA-C*05 on the cell surface. Collectively, these data provide insight into how the α1/α2 domains and the peptide-binding cleft of HLA-C molecules can not only have a direct influence on HLA stability and peptide repertoire, but also influence cell surface expression levels.HLA-C alleles, which we show influences HLA-C expression levels, we performed sequence alignments of the exons 2 and 3 region of HLA-C alleles with the available non-human primate MHC-C alleles, and inferred their phylogenetic relationship  or tapasin have been proposed to contribute towards lower surface expression of HLA-C in comparison to HLA-A and HLA-B2425Studies of chicken MHC have reported an inverse correlation between diversity of peptide repertoire and cell surface MHC class I expression, with low expression correlating with resistance to Marek’s disease37373839C*07-like alleles has been maintained in primates for millions of years and can be found in modern populations of chimpanzees and other species, while no C*05-like alleles were found in the chimpanzee sequences available. By contrast, the 3′ miRNA binding site polymorphism seems to have arisen since the split of the human and chimpanzee ancestors, through a gene conversion event from an HLA-B sequenceOur results, combined with previous work, show that HLA-C expression is modulated by multiple factors acting at several levels, from transcription to miRNA binding and peptide selectivity mediated by the antigen-binding cleft—consequently leading to a net effect that determines abundance at the cell surface . Such diC*05 and C*07 hybrid constructs were made by amplifying∼2.04 and 2.06 kb genomic fragments of HLA-C*05:01:01:01 and HLA-C*07:02:01:03 respectively, which contained 776 or 766 bp of the respective HLA 5′UTR and exons 1–3 up to a midpoint in intron 3, which was fused to a∼3.58 kb fragment of the genomic H-2Kb gene, beginning at a midpoint in intron 3 and containing exons 4–8 and the H-2Kb 3′UTR. For experiments with swapped promoters/5′UTR, additional hybrid constructs were made where the 5′ flanking region of the HLA genes was interchanged, such that the HLA-C*05:01:01:01 promoter/5′UTR was fused to the exons 1–3 sequence of HLA-C*07:02:01:03, and vice versa. The region from the genomic H-2Kb gene was the same as described above. These hybrid constructs were transfected into 721.221 cells using optimized electroporation conditions  using a Genepulser II (Bio-Rad). A limiting concentration of the pmax-GFP plasmid (Lonza) was co-transfected as a transfection control. The cells were collected 48 h post-transfection, and used for flow cytometry or RNA isolation/QPCR experiments.The HLA-C*05:01:01:01 (776 bp upstream of the start codon) or HLA-C*07:02:01:03 (766 bp upstream of the start codon) genes were cloned into a luciferase containing pGL4.14 (Promega) basic promoter-less vector. HEK 293 T or 721.221 cells were transfected with the luciferase constructs containing the 5′UTR/promoter from either C*05, C*07 or no promoter, along with the co-transfection of the Renilla luciferase vector, pGL4.74 (Promega), using TransIT 2020 transfection reagent (for HEK 293T cells) or optimized electroporation conditions (for 721.221 cells). Cells were lysed after 24 h (HEK 293T) or 5 h (721.221) post-transfection, and firefly and renilla luciferase activities were measured using dual luciferase reporter assay system (Promega) and the Glomax multi detection system (Promega). The firefly luciferase activity was normalized relative to Renilla luciferase for each transfection, and the luciferase activity of each reporter construct was calculated as a fold change relative to the activity of pGL4.14-basic vector lacking a promoter.The promoters/5′UTR of C*05 and C*07 lentiviral expression constructs were made by amplifying the cDNA of HLA-C*05:01:01:01 and HLA-C*07:02:01:03 genes from exons 1–8, and cloning it into the pHRsinUbEm expression plasmid , with the inclusion of the HA tag at the N-terminus of C*05 and C*07, just after the signal peptide sequence. For the modified C*05 and C*07 lentiviral expression constructs, the cDNA of exons 1–3 from the respective HLA genes was fused to the cDNA of exons 4–8 of the murine H-2Kb gene, and cloned into the pHRsinUbEm expression plasmid, with inclusion of the N-terminal HA tag. The lentiviral HLA expression plasmids were co-transfected with the vesicular stomatitis virus-G envelope plasmid pMD2.G (Addgene) and packaging plasmid psPAX2 (Addgene), containing HIV-1 Gag and Rev, into HEK 293T cells to package lentiviral particles. Viral titres were determined by serial dilution and transduction of HEK 293T cells. 721.221 cells were transduced with the packaged lentivirus at a multiplicity of infection of 20, in the presence of polybrene (Santa Cruz Biotechnology), added at a final concentration of 8 μg ml−1. Cells were collected 72 h post transduction and flow cytometry, RNA isolation/QPCR or immunoblotting experiments were performed.The HLA-C cell surface expression was measured in transfected or transduced cells using alexa fluor 647 anti-human HLA-A, B, C antibody  or anti-HA.11 antibody  along with allophycocyanin goat anti-mouse Ig . HLA-C or HA staining was determined on GFP+ cells. For intracellular staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences), followed by staining using phycoerythrin anti-human HLA-A, B, C antibody  or anti-HA.11 antibody  along with allophycocyanin goat anti-mouse Ig . HLA staining using the same antibodies on non-transfected cells was used as a negative control. Data obtained were analysed using the FlowJo software (Tree Star).H-2Kb gene (which formed a common region of the HLA-C constructs), GFP , Copgreen (for detecting GFP in pmaxGFP) and UBC. The sequences of the primers used for these assays were as follows:Total RNA was isolated from cells using the RNeasy isolation kit (Qiagen), and cDNA prepared using the Quantitech Reverse Transcription kit (Qiagen). SYBR-green based quantitative PCR assays were designed and optimized for the H2Kb: 5′-GAGCTGCAATAGTCACTGGAG-3′ and 5′-CCATCACTTTACAATCTGGGAGAG-3′;GFP: 5′-AAGAACGGCATCAAGGTGAAC-3′ and 5′-CAGGTAGTGGTTGTCGGG-3′;Copgreen: 5′-AGGACAGCGTGATCTTCACC-3′ and 5′-CTTGAAGTGCATGTGGCTGT-3′;UBC: 5′-AACATCCAGAAAGAGTCCACC-3′ and 5′- CTTGTCTTGGATCTTTGCCTTG-3′.−dCt, where dCt is the difference of the cycle threshold between the transcript of the gene of interest and the reference gene transcript.Standard curves for each of the assays were performed using serial dilutions of cDNA and amplification efficiencies were determined. Relative expression was expressed as 2Whole cell lysates were prepared in NP-40 lysis buffer containing 150 mM NaCl, 1% NP-40 , 50 mM Tris pH 8.0, supplemented with protease inhibitors (Roche). Protein concentration of cell lysates was determined using Pierce BCA Protein Assay Kit (Thermo Scientific) and equal amount of protein was loaded onto denaturing polyacrylamide gels. HA-tagged HLA-C protein was visualized using anti-HA.11  and normalized using anti-GFP (Life technologies) and anti-GAPDH  antibodies. Membranes were stained with IRDye 800CW goat-anti-mouse IgG and IRDye 680LT goat-anti-rabbit IgG secondary antibodies and visualized and quantified using an Odyssey Infra-Red Imaging System (LI-COR Biosciences).U-tests were used for comparing two experimental groups, with a 5% significance level. For swapped promoter analyses, a Bonferroni correction for multiple testing was used; considering P=0.05 for four independent hypotheses, the significance threshold used for this analysis was P=0.0125.Statistical tests were performed using GraphPad Prism and non-parametric Mann–Whitney Escherichia coli as inclusion bodies as previously describedSoluble class I heterodimers of HLA-C*05 and HLA-C*07 heavy chain and full-length β2-microglobulin (β2m) were expressed in −1 in 10 mM Tris-HCl pH 8, 150 mM NaCl using 1.8 M Na malonate pH 7. The HLA-C*05:01-SAE crystals were flash frozen in liquid nitrogen. Crystals of HLA-C*0702-RYR were grown in 0.1 M HEPES pH 8.5, 2 mM ZnSO4 and 28% jeffamine ED-2001 (Hampton), using the same technique as for the HLA-C*05-SAE crystals. The HLA-C*07:02-RYR crystals were soaked in a mother liquid solution with the addition of 25% ethylene glycol prior to be flash frozen in liquid nitrogen. The data were collected on the MX1 beamline at the Australian SynchrotronCrystals of the HLA-C*05-SAE were grown by the hanging-drop, vapour-diffusion method at 20 °C with a protein/reservoir drop ratio of 1:1, at a concentration of 3 mg ml9 721.221 cells for each HLA-C using the pan class I antibody W6/32 immobilized and cross-linked to protein A resin. Captured HLA-peptide complexes were eluted with 10% acetic acid. The dissociated complexes were further separated by Reversed-phase HPLC to isolate and fractionate the bound peptides before analysis with a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific)The HLA class-I negative 721.221 cells stably transfected with HLA-C*05:01 or HLA-C*07:02 were utilized to obtain the peptide repertoires in a previously described manner53HLA-C and chimpanzee MHC-C alleles were obtained from the IMGT databaseC*05 and C*07 sequences. Sequences were aligned with MUSCLE (v3.8.31) (ref. Nucleotide sequences for 31) ref.  and the 31) ref.  was usedHLA-C*05:01:01:01 and HLA-C*07:02:01:03 were identified in the NCBI nucleotide database using BLAST+ (v. 2.3.0)  length was 798 bases for the alignment, which included 792 bp for C*05 and 796 bp for C*07; the 3′UTR region included 541 bp after the stop codon in C*05 and C*07. For each complete sequence, we determined the HLA-C allele by aligning the individual sequence in the HLA-C IMGT data setHLA-C allele and HLA-C*05 and HLA-C*07 in a phylogenetic tree, we calculated a metric quantifying the phylogenetic distance between the query HLA allele and its relative distance to HLA-C*05 and HLA-C*07 in the specific tree. This was done in the following way: phylogenetic distances as branch lengths from the HLA-C allele to HLA-C*05 and to HLA-C*07 were extracted using the R package apeHLA-C sequence to HLA-C*05 divided by the sum of the distances of the query HLA-C sequence to HLA-C*05 and HLA-C*07.Sequences homologous to .0) ref. . SequencStructural information has been deposited in the Protein Data BankHow to cite this article: Kaur, G. et al. Structural and regulatory diversity shape HLA-C protein expression levels. Nat. Commun.8, 15924 doi: 10.1038/ncomms15924 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Systematic investigation was carried out to disclose the effect of PC71BM on the blend morphology and charge transport behavior. It is found that the PC71BM tends to intermix with the PBDB-T donor compared to the ITIC counterpart. A small amount of PC71BM in the ternary blend is helpful for ITIC to aggregate and form efficient electron-transport pathways. Accordingly, the electron mobility is increased and the density of electron traps is decreased in the ternary blend in comparison with the PBDB-T:ITIC blend. Finally, the suppressed bimolecular recombination and enhanced charge collection lead to high PCE for the ternary solar cells.The development of non-fullerene acceptor molecules have remarkably boosted power conversion efficiency (PCE) of polymer solar cells (PSCs) due to the improved spectral coverage and reduced energy loss. An introduction of fullerene molecules into the non-fullerene acceptor-based blend may further improve the photovoltaic performance of the resultant ternary PSCs. However, the underlying mechanism is still debatable. Herein, the ternary PSCs based on PBDB-T:ITIC:PC For example, Bo et al. have reported high-performance ternary PSCs employing non-fullerene and fullerene acceptors simultaneously for the first time, in which they found that a small amount of fullerene is in favor of boosting the photovoltaic performance of the devices  in recent years. The power conversion efficiencies (PCE) of PSCs employing non-fullerene acceptors have increased rapidly as compared to the PSCs using fullerene derivatives as acceptors  is used as the third component to prepare the ternary PSCs. The optimized PSCs based on ternary PBDB-T:ITIC:PC71BM blend demonstrate a higher PCE of 10.2% than 9.2% of the binary PBDB-T:ITIC devices and 8.1% of the PBDB-T:PC71BM PSCs. Further studies are mainly focused on the effect of PC71BM on the blend morphology and charge transport behavior. It is found that the PC71BM tends to intermix with the PBDB-T donor compared to the ITIC counterpart. A small amount of PC71BM in the ternary blend is helpful for ITIC to aggregate and form efficient electron-transport pathways. Accordingly, the electron mobility is increased and the density of electron traps is decreased in the ternary blend in comparison with the PBDB-T:ITIC blend. Finally, the suppressed bimolecular recombination and enhanced charge collection lead to an enhanced PCE for the ternary solar cells.Herein, a reported wide bandgap polymer poly dithiophene))-alt- benzodithiophene-4,8-dione))] (PBDB-T) is used as the donor, organic molecule -indanone))- 5,5,11,11-tetrakis(4-hexylphenyl)-dithieno-s-indaceno dithiophene) (ITIC) is used as the acceptor and -phenyl-C71-butyric acid methyl ester . The EQE data were measured by solar cell spectral response measurement system . The film absorption and fluorescence spectra were recorded on Agilent Cary 60UV-Vis spectrophotometer and Perkin-Elmer LS 55 spectrofluorometer, respectively. The thicknesses of individual layers were measured with a surface profilometer. The 2D-GIXD data were acquired at station 14B in Shanghai Synchrotron Radiation Facility.The current density-voltage (J-V) curves of the PSCs were traced by a computer-controlled Keithley 2400 Source Meter under simulated solar light illumination  blend were firstly evaluated. The ratio of A1:A2 is varied from 1:0 to 0:1 while the ratio of D:(A1+A2) is fixed at 1:1 in order to keep a constant active layer thickness. The detailed photovoltaic parameters of the resultant PSCs with different ratio of two acceptors are listed in Table OC, JSC, FF, and PCE on the PC71BM content for the resultant PSCs are plotted in Figure OC, it undergoes gradually decrease from 0.902 V for the binary PBDB-T:ITIC PSCs to 0.856 V for the binary PBDB-T: PC71BM PSCs with increasing PC71BM content from 0 to 1 in the ternary blend PSCs. The change is reasonable since the LUMO of ITIC is higher than that of PC71BM, and the electron transport and collection will occur in PC71BM phases when the PC71BM amount is larger. The interesting thing is that the JSC are initially increased and then decreased with the increase of PC71BM contents for the resultant ternary PSCs. The FF shows similar trend. Considering the intrinsic absorption properties between ITIC and PC71BM, the substitution of small amount of ITIC by PC71BM would decrease the total absorption of the ternary PBDB-T:ITIC:PC71BM blend active layer. Thus, the improved JSC and FF for the ternary PBDB-T:ITIC:PC71BM PSCs may imply that both the exciton dissociation and charge-collection efficiencies are enhanced in comparison to the binary PBDB-T:ITIC PSCs. It is plotted the illuminated J-V curves of the PBDB-T:ITIC PSCs, the PBDB-T:PC71BM PSCs and the optimized ternary PBDB-T:ITIC:PC71BM PSCs in Figure OC of 0.902 V, a JSC of 15.06 mA/cm2, a FF of 0.69, respectively, leading to a PCE of 9.38%. Due to the limited spectral coverage, the PBDB-T:PC71BM PSCs show a lower JSC of 13.64 mA/cm2, and finally result in a PCE of 8.21% together with a VOC of 0.856 V and a FF of 0.703. The ternary PBDB-T:ITIC:PC71BM (1:0.8:0.2) PSCs demonstrate a VOC of 0.892 V, a JSC of 15.98 mA/cm2, a FF of 0.717 and an overall PCE of 10.22%. Although the VOC is a little lowered in comparison to the binary PBDB-T:ITIC PSCs, the increased JSC and FF boost the PCE enhancement of the ternary PSCs. The external quantum efficiency (EQE) curves of the three devices are shown in Figure 71BM PSCs cover at a range of 300–700 nm with EQE higher than 70% at 450–650 nm. The PBDB-T:ITIC PSCs show extended spectral coverage of 300–800 nm due to the narrow bandgap of ITIC. The ternary PBDB-T:ITIC:PC71BM PSCs show similar spectral response profile but high EQE compared to the PBDB-T:ITIC PSCs. The internal quantum efficiency (IQE) of these devices are also measured to clarify the absolute quantum efficiency in these devices and the curves are plotted in Figure 71BM into the PBDB-T:ITIC blend. The absorption spectra of the active layers in three kinds of PSCs are plotted in Figure 71BM (1:0.8:0.2) active layer shows a little low absorption at 550–750 nm due to decreased ITIC content compared to the PBDB-T:ITIC (1:1) film. Although the PBDB-T:PC71BM (1:1) film demonstrates enhanced absorption at 300–500 nm, its absorption at 550–700 nm is remarkably decreased. Photoluminescence (PL) quenching experiments were carried out to check the charge transfer status in these films. As shown in Figure 71BM favors to quench the excitons dominated on the PBDB-T donor.The photovoltaic performance of the PSCs based on the PBDB-T:ITIC:PCph) and effective voltage (Veff) is investigated to judge the charge generation and collection status in the PSCs based on the PBDB-T:ITIC (1:1), PBDB-T:ITIC:PC71BM (1:0.8:0.2), and PBDB-T:PC71BM (1:1) blends , PBDB-T:ITIC:PC71BM (1:0.8:0.2), and PBDB-T:PC71BM (1:1) blends. The device based on ternary PBDB-T:ITIC:PC71BM (1:0.8:0.2) blend demonstrates the highest saturation current, indicating its highest charge-generation capability. Charge dissociation probability P is defined as Jph/Jsat. When a high bias is applied, the charge recombination is suppressed and most of photo-generated charges are extracted, leading to P close to100%. It is calculated that the P values under short-circuit condition (Vappl = 0 V) are 90.6, 91.9, and 94.9%, respectively, for the PSCs based on the PBDB-T:ITIC (1:1), PBDB-T:ITIC:PC71BM (1:0.8:0.2), and PBDB-T:PC71BM (1:1) blends. The PBDB-T:PC71BM (1:1) device shows the best charge-extraction capability, whereas the charge-extraction ability is a little poor for the PBDB-T:ITIC (1:1) device. The ternary PBDB-T:ITIC:PC71BM (1:0.8:0.2) shows improved exciton dissociation and charge extraction compared to the PBDB-T:ITIC (1:1) device. The charge recombination status in these devices is also investigated via the dependence of JSC on the incident light intensity as shown in Figure SC is dependent on the incident light intensity with JSC∝Iα, in which exponential factor α would be close to unity without bimolecular recombination  device has the highest α value of 0.964, whereas α value of the PBDB-T:ITIC (1:1) device is merely 0.935 implying severe bimolecular recombination within the film. Such kind of bimolecular recombination in the PBDB-T:ITIC (1:1) device is to some extent suppressed with α value of 0.955 in ternary PBDB-T:ITIC:PC71BM (1:0.8:0.2) device.The relationship between photocurrent  method  blend film shows relatively high electron and hole mobility of 8.45 × 10−4 and 5.30 × 10−4 cm2V−1s−1, respectively. However, the electron and hole mobility of the PBDB-T:ITIC (1:1) film is 3.05 × 10−4 and 2.70 × 10−4 cm2V−1s−1. After incorporating PC71BM, the electron and hole mobility of the resultant PBDB-T:ITIC:PC71BM (1:0.8:0.2) film is increased to 4.55 × 10−4 and 3.37 × 10−4 cm2V−1s−1. As it is known, the high and balanced charge transport may help to efficient charge extraction and hence low charge recombination. This is in agreement with the recombination status in these PSCs. It is speculated that the incorporation of PC71BM improves the PBDB-T and ITIC interpenetrating networks and thus the photovoltaic performance.As discussed above, with the introduction of PC71BM (1:0.8:0.2), and PBDB-T:PC71BM (1:1) blend films are investigated and their AFM and TEM images are shown in Figures xy = 0.29 Å−1 in the in-plane direction, attributing to its strong (100) diffraction. The pure ITIC film demonstrates two featured peaks at qz = 0.26 Å−1 and qz = 0.53 Å−1 in out-of-plane direction and one peak in in-plane direction at qxy = 0.36 Å−1. 2D-GIXD patterns and line-cut profiles of the PBDB-T:ITIC (1:1), PBDB-T:ITIC:PC71BM (1:0.8:0.2) and PBDB-T:PC71BM (1:1) films are shown in Figure 71BM (1:1) films in contrast to the pure PBDB-T film. However, the diffraction signal originated from the ITIC component is not obviously presented in PBDB-T:ITIC blend film, implying its poor aggregation. In the PBDB-T:ITIC:PC71BM (1:0.8:0.2) blend film, the diffraction signal from PBDB-T is still presented but weakened. More importantly, two additional peaks marked 1 and 2 are presented in the ternary blend film compared to the PBDB-T:ITIC (1:1) counterpart, which corresponds to the featured diffraction from ITIC in the in-plane and out-of-plane directions. Bo et al. observed similar phenomenon in their work  blend film though the ITIC content is decreased. In other words, the small amount of PC71BM may serve as “lubricant” to favor ITIC molecules to aggregate out of the polymer PBDB-T matrix.The morphology of the PBDB-T:ITIC (1:1), PBDB-T:ITIC:PC71BM should be better than that between PBDB-T and ITIC. The electron mobility of the PBDB-T:ITIC (1:x) and PBDB-T:PC71BM (1:x) blend films are measured and its dependence on the acceptor ratio is plotted in Figure −5 cm2V−1s−1. Both the as-prepared film deposited in CB solution and the annealed film deposited in CB:DIO  solution are tested. The results show that the continuous electron-transport tunnels is formed in the PBDB-T:ITIC film with the ITIC/PBDB-T ratio <20%. The DIO additive and thermal annealing donot change the electron transport remarkably. For the as-prepared PBDB-T:PC71BM film, the critical PC71BM content is increased to 25%. The DIO additive and thermal annealing lowers the critical PC71BM content. This indicates that the PBDB-T is inclined to intermix with PC71BM better than with ITIC. In the ternary PBDB-T:ITIC:PC71BM (1:0.8:0.2) blend film, the small amount of PC71BM may mix with PBDB-T and does not form networks. Moreover, the existence of PC71BM favors ITIC aggregating to form electron-transporting networks. This is confirmed by the increased electron mobility and the XRD results compared to the PBDB-T:ITIC (1:1) film.The prerequisite to get such a conclusion is that the compatibility between PBDB-T and PC71BM (1:0.8:0.2) and PBDB-T:PC71BM (1:1) blend shown in Figure TFL is the trap-filled limit voltage, e is the elementary charge of electron, L is the thickness of film, ϵ0 is the vacuum permittivity, and ϵr is the relative dielectric constant (ϵr = 3). The VTFL of three devices is 0.12, 0.05, and 0.02 V. The calculated deep trap density is 3.3 × 1015, 1.5 × 1015, and 7.3 × 1014 cm−3, respectively. It confirms that the incorporation of PC71BM is helpful for ITIC molecules to move out of PBDB-T phases and thus the density of electron traps is reduced. The increased electron mobility and the decreased electron traps are helpful to enhance the photovoltaic performance of the PBDB-T:ITIC:PC71BM (1:0.8:0.2).The electron trap-state density in these blend films is further investigated. From the I–V curves of the electron-only devices based on the PBDB-T:ITIC (1:1), PBDB-T:ITIC:PC71BM (1:0.8:0.2) blend were fabricated and its PCE was increased to 10.2% compared to 9.2% for the PBDB-T:ITIC (1:1) devices. The mechanism accounting for the enhanced photovoltaic performance is discussed in detail. It is found that the PC71BM tends to intermix with the PBDB-T donor compared to the ITIC counterpart. A small amount of PC71BM in the ternary blend is helpful for ITIC to aggregate and form efficient electron-transport pathways. The electron mobility is increased and the density of electron traps is decreased in the ternary PBDB-T:ITIC:PC71BM (1:0.8:0.2) blend in comparison with the PBDB-T:ITIC blend. Finally, the suppressed bimolecular recombination and enhanced charge collection lead to a high PCE for the ternary solar cells.In summary, the ternary PSCs based on PBDB-T:ITIC:PCBW and CY conceived and designed the experiments. BW performed the experiments. RZ performed 2D-GIXD measurements. QY performed the absorption and PL experiments. BW and YF analyzed data. BW wrote the manuscript. All authors discussed and commented on the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oryza sativa L.) varieties, most of which contain submergence-inducible SUB1A on the polygenic SUBMERGENCE-1 (SUB1) locus. It has been shown that the SUB1 locus encodes two Ethylene-Responsive Factor (ERF) genes, SUB1B and SUB1C, in all O. sativa varieties. These genes were also found in O rufipogon and O nivara, wild relatives of O. sativa. However, detailed analysis of the polygenic locus in other Oryza species has not yet been made.Tolerance to complete submergence is recognized in a limited number of Asian rice  contains supplementary material, which is available to authorized users. Oryza sativa L.), many flood-prone lands are used for rice production, reason why so many studies are developed aiming to understand and overcome flooding response mechanisms.Abiotic stresses such as flooding can prevent plants from attaining their full genetic potential for growth and reproduction. In Asia, the leading producers of rice (SUBMERGENCE-1 (SUB1) is responsible for conferring submergence tolerance to the rice landrace Flood Resistant 13A (FR13A). Detailed sequence analysis revealed that the locus encodes a variable cluster of up to three Ethylene-Responsive Factors (ERF): SUB1A, SUB1B and SUB1C  called O. sativa varieties contain SUB1B and SUB1C, whereas SUB1A is present only in a limited number of accessions. Genotypes containing submergence-inducible SUB1A restricts underwater ethylene production, which maintains mRNA and protein accumulation of gibberellic acid (GA) signaling repressors during submergence  consortium , O. sativa L. ssp japonica (AA), O. rufipogon Griff. (AA), O. nivara Sharma et Shastry (AA), O. glumaepatula Steud. (AA), O. glaberrima S. (AA), O. barthii A. Chev. (AA), O. meridionalis Ng. (AA), O. punctata Kotschy ex Steud. (BB), and O. brachyantha Chev. et Roehr (FF).Wild rice species generally lack agriculturally important traits. However, they carry many desirable genes that have been lost in cultivated rice. Here, we report that previous analyses made by Fukao et al.  and NiroSUB1-like genes , O. nivara (AA), O. glumaepatula (AA), O. glaberrima (AA), O. sativa (AA), and O. punctata (BB) contained both SUB1B- and SUB1C-like genes side by side on chromosome 9 (Chr 9), as shown in Fig. O. sativa (AA) , "SUB1-like Chr 6" (SUB1chr6) and "SUB1-like Chr 9" (SUB1chr9) , which is the SUB1chr2 gene found in O. sativa is already known to interact with a Myc transcription factor called OsBP-5, coregulating the expression of the waxy gene  and O. barthii (OBART01G06430), respectively. To identify possible translocation events related to these specific loci we aligned regions corresponding to 50 Kb upstream and 50 Kb downstream of ONIVA01G39120 and OBART01G06430 to SUB1 loci (Chr 9) of each Oryza species. The alignments show no similar regions when SUB1 loci are compared to ONIVA01G39120, nevertheless we can identify an orthologous region (~8.5 Kb) when SUB1 loci of all genome AA species are compared to OBART01G06430 , O. barthii (AA) and O. brachyantha (FF) encoded only one SUB1-like gene, all of them more related to SUB1B than to SUB1C.It is also interesting to highlight that the genomes of SUB1 locus were found in Chr 9 of Leersia perrieri (A. Camus) Launert.  demonstrating the presence of duplication events in the onset of SUB1B and SUB1C, which occurred after the divergence of Oryzeae from other grasses. This duplication event seems to have occurred before the speciation of L. perrieri.Three genes closely related to SUB1A-like genes were not detected in the genomes of these rice relatives, except for O. nivara  and original SUB1A-1 demonstrates that, despite differences in the C-terminal region, the two O. nivara sequences are probably variants  from the same locus. O. brachyantha also presented a gene that was similar to SUB1 (OB09G12530), but the absence of a serine at position 13, and other differences along their sequence prevented its classification as a SUB1A-like gene. It is interesting to notice that the SUB1A-like genes found in O. nivara (ONIVA01G39120) and O. barthii (OBART01G06430), were not on Chr 9, but on Chr 1.ara Fig. . An aligara Fig.   was calculated  for SUB1C-like genes. The freedom that SUB1B-like genes have to be modified may have been essential to the emergence of SUB1A-like genes, which are extremely close to SUB1B genes.To estimate how natural selection is acting on the coding sequences of SUB1A-like genes shows that the gene found in O. nivara must have a similar regulation to the SUB1A-1 allele responsible for tolerance to submergence in O. sativa  and SUB1A-2 (non-upregulated by submergence) alleles were found. Now we should further investigate the possibly tolerant phenotype resulting from the introgression of ONIVA01G39120 into O. sativa elite cultivars and how is it regulated since this constitutes a very relevant information to breeders.The analysis of the upstream sequences  of SUB1-like genes isolated from these rice relatives . However, our gene structure analysis based on RNAseq data did not detect 3' UTR sequences of SUB1C-like genes (Additional file SUB1C-like genes was sought within 1.5 kb downstream of the stop codon in these genes and regions highly similar to the O. sativa intron were found in ORUFI09G04400, ONIVA09G04180, OGLUM09G04680, OGLAB09G030300 and OPUNC09G03530 indicating that most SUB1C-like genes identified in Oryza species possess traces of an intron in their terminal region (Additional file SUB1-like genes identified in wild rice species through genome-wide sequence analysis. Our chromosomal location, phylogenetic, and gene structure analyses have revealed that the SUB1 locus is conserved in the long arm of Chr 9 in most Oryza species with AA-, BB- and FF-genomes. We also showed that the SUB1A-like gene of O. nivara is on Chr 1 and that L. perrieri, a grass-tolerant deep-flooding, presents three ERF genes in SUB1 locus, similar to that found in flooding tolerant rice. This finding raises the possibility that an association of this gene with flooding tolerance in L. perrieri exists. Along with genes found in Chr 1, SUB1-like genes found in Chr 2 and Chr 6 represent some new interesting information. These genes should now be further investigated in order to check their biological and agricultural significance. Allelic survey of diverse ecotypes in wild rice species is necessary to determine the evolutionary origin and biological significance of SUB1A in the genus Oryza (Additional file In summary, here we present