The viral envelope glycoprotein (Env) is the major target for antibody (Ab)-mediated vaccine development against the Human Immunodeficiency Virus type 1 (HIV-1). Although several recombinant Env antigens have been evaluated in clinical trials, only the surface glycoprotein, gp120,  used in the ALVAC prime-AIDSVAX gp120 boost RV144 Phase III HIV vaccine trial was shown to contribute to protective efficacy, although modest and short-lived. Hence, for clinical trials in southern Africa, a bivalent protein boost of HIV-1 subtype C gp120 antigens composed of two complementary gp120s, from the TV1.C (chronic) and 1086.C (transmitted founder) HIV-1 strains, was selected. Stable Chinese Hamster Cell (CHO) cell lines expressing these gp120s were generated, scalable purification methods were developed, and a detailed analytical analysis of the purified proteins was conducted that showed differences and complementarity in the antigenicity, glycan occupancy, and glycan content of the two gp120 molecules. Moreover, mass spectrometry revealed some disulfide heterogeneity in the expressed proteins, particularly in V1V2-C1 region and most prominently in the TV1 gp120 dimers. These dimers not only lacked binding to certain key CD4 binding site (CD4bs) and V1V2 epitope-directed ligands but also elicited reduced Ab responses directed to those epitopes, in contrast to monomeric gp120, following immunization of rabbits. Both monomeric and dimeric gp120s elicited similarly high titer Tier 1 neutralizing Abs as measured in standard virus neutralization assays. These results provide support for clinical evaluations of bivalent preparations of purified monomeric TV1.C and 1086.C gp120 proteins. HIV-1 infection and acquired immunodeficiency syndrome (AIDS) represent a major public health concern. HIV/AIDS is most prevalent in sub-Saharan Africa, where almost 70% of all HIV-infected people live. HIV-1 subtype C accounts for over 95% of infections in southern African . While rDiverse HIV vaccines have been tested in early phase clinical trials . The earThe first evidence of HIV vaccine efficacy came from the RV144 Phase 3 trial in Thailand . This trThe Pox-Protein Public Private Partnership or “P5” was formed in 2010 to follow-up on the clinical results of RV144 . The P5 The TV1.C and 1086.C gp120 antigens were selected in consultation with a group of HIV vaccine experts to provide a bivalent subtype C protein boost component. Here we report the generation of stable CHO cell lines expressing these two gp120s, development of a scalable purification process, a comprehensive analytical characterization of the purified gp120s, and confirmation of the immunogenicity of the candidate proteins. These studies serve as a foundation for cGMP manufacture of these candidates for post-RV144 clinical evaluations.Reagents are listed in Methods used to generate and evaluate stable cell lines expressing gp120 are provided in 3—column (cation exchange) and DEAE (anion exchange) column. Details are described in Initially, gp120 proteins were purified using a three-step purification process involving Galanthus Nivalis-Agarose (GNA) affinity column, anion-exchange DEAE column and a final ceramic hydroxyapatite (CHAP) column, as described previously . SubsequThe gp120 mutants  used in serum-mapping studies were generated as described previously .Binding affinities of TV1.C and 1086.C gp120 proteins to a gp120 ligands were determined using Surface Plasmon Resonance . Approximately 200 RU of sCD4 or mAbs were immobilized directly onto a CM5 sensor chip via amine coupling. Varying concentrations of gp120, either TV1.C or 1086.C, were then injected at 100 μl/min and regenerated using either two 60s-injections of Glycine-HCl pH 3.0 or 10mM NaOH pH 10.0. The binding studies were performed at 25°C with PBS (+0.1% BSA) as running buffer. The experimental curves were fitted to a 1:1 Langmuir binding model using BIAevaluation software 3.2 (BIAcore Inc). Binding of TV1.C or 1086.C gp120s to 34 HIV+ sera from South African volunteers was performed using a D7324-capture ELISA, as reported previously .Glycopeptide analyses of TV1.C or 1086.C gp120 monomer samples (~75 μg protein) were performed on partially deglycosylated and non-deglycosylated samples. Samples were partially deglycosylated with endoglycosidase H (EndoH). For partial deglycosylation, samples were denatured with 2 M urea followed by the addition of 2 μL of Endo H (≥5 units/mL) and incubated for 48 hrs at 37°C. Partially deglycosylated and non-deglycosylated samples were denatured with 6 M urea in 50 mM Tris buffer (pH 8.0) containing 3 mM EDTA and were fully reduced using 5 mM TCEP at room temperature (RT) for 1 hr. Following reduction, samples were alkylated with 20 mM IAM at RT for 1 hr in the dark. Excess IAM was quenched with DTT to a final concentration of 25 mM for 20 min at RT. The reduced and alkylated samples were buffer exchanged and concentrated using a 30 kDa MWCO filter  prior to trypsin digestion. Samples were subsequently digested with trypsin (50:1 protein: enzyme ratio) at 37°C and incubated overnight, followed by second trypsin addition under the same conditions. Resulting gp120 digests were subjected to LC/MS/MS analysis using high and low resolution mass spectrometers described in the next paragraph. Glycopeptide identification and analysis of glycosylation site occupancy were performed as described previously .2O + 0.1% formic acid and solvent B: 99.9% CH3CN + 0.1% formic acid and a C18 PepMap 300™ column . Approximately 5 μl of sample was injected into the column at a flow rate of 5 μl/min using the following gradient: a linear increase to 40% B in 50 minutes then to 90% B in 10 min. The column was held at 90% B for 10 min before re-equilibration to starting conditions. Data were collected in a data dependent acquisition mode in which the five most intense ions in a high resolution survey scan in the FTICR cell were sequentially and dynamically selected for subsequent collision-induced dissociation (CID) in the LTQ linear ion trap. The LTQ Velos mass spectrometer was set up to perform experiments by alternating CID and ETD acquisition. Data dependent acquisition (DDA) was set up to acquire 10 scan events: for every one full MS scan in the mass range, 300–2000 m/z, each selected m/z in the MS scan were subjected to three MS/MS events- (a) CID, (b) ETD, and (c) CID of the charge reduced precursor in the previous ETD event. The mass spectrometric parameters used for the experiment were: spray voltage 3.0 kV, S-lens value between 45–55%, capillary temperature of 250°C, normalized collision energy of 35% for CID, and the ion-ion reaction for ETD between the precursor ion and the radical anion, fluoranthane, was set at AGC target value of 2x105 and 100 msec ion-ion reaction time. To improve ETD efficiency, supplemental activation was turned on.Disulfide bond patterns of the TV1.C and 1086.C gp120 were determined by mapping the disulfide linked peptides with mass spectrometry. Samples containing 75 μg of gp120 protein were alkylated with 10-fold molar excess of 4-vinylpyridine in the dark for an hour at room temperature to cap free cysteines. Alkylated samples were then deglycosylated with 1 μL PNGase F solution  at 37°C for a week. Fully deglycosylated alkylated samples were digested with trypsin (protein to enzyme ratio of 30) overnight. To ensure reproducibility, deglycosylation and subsequent trypsin digestion were performed at least two times. Following tryptic digestion, samples were analyzed using a hybrid linear ion trap Fourier Transform-Ion Cyclotron Resonance  mass spectrometer coupled with a nanoACQUITY Ultra Performance Liquid Chromatography (UPLC) system  for high resolution LC/MS/MS analysis and an LTQ Velos mass spectrometer  equipped with electron transfer dissociation (ETD) and a ACQUITY UPLC system for low resolution LC/MS/MS analysis. Chromatographic separation for both low and high resolution LC/MS/MS analyses was performed using mobile phases consisted of solvent A: 99.9% deionized HImmunization studies were conducted at Josman LLC , a USDA licensed research facility (No. 93-R-0260) with a Public Health Service (PHS) Assurance from the NIH (No. A3404-01). Five young adult female New Zealand White rabbits were used in each study group. Rabbits were immunized with gp120 proteins using MF59 plus carbopol as previously described . Three i4SCN) displacement ELISA. Both assays employed the matched gp120 Ags and were performed as previously described [Env-specific binding Ab titers were measured by a standard ELISA assay, and Ab avidity indices were measured using an ammonium thiocyanate ) as reported previously –30 and sTo determine epitope-specific Abs in 2wp3 sera from rabbits immunized with gp120 proteins, WT or mutant gp120s were captured onto the ELISA plates using D7324 as previously described . The bouPercent epitope-directed binding was calculated as the (EC50 of binding to the mutant version of gp120 divided by EC50 of binding to the WT gp120) x 100.Comparisons among groups were carried out using analysis of variance (1 way ANOVA). A Kruskal-Wallis test was used to analyze differences between multiple epitope-directed groups. For all comparisons, a two-sided p<0.05 was considered statistically significant.The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.In addition, new names contained in this work have been submitted to IPNI, from where they will be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix In consultation with experts in the HIV vaccine field and based on information available at the time, a list of criteria were established to guide the selection of two gp120 proteins as a boost for future clinical trials for the southern African region . Two gp1Stable CHO cell lines expressing gp120s from TV1.C and 1086.C were generated after screening a large numbers of clones . The pre3 resin followed by differential elution of gp120 monomers and dimers using a pH gradient wash. Further polishing on DEAE anion exchange column in flow through mode resulted in highly pure monomeric gp120 protein (>95% purity and >90% monomer content). The 2-step anion exchange purification method was used for the production of proteins analyzed as described in the following sections.During the initial screening phase, gp120 proteins were purified using GNA  affinity chromatography followed by polishing with DEAE and CHAP columns . SDS-PAGThe glycosylation site occupancy and composition of the CHO-expressed TV1.C and 1086.C gp120 monomers was assessed by mass spectrometry (MS) analysis of partially de-glycosylated and non-deglycosylated samples . The tryFor TV1.C gp120, 9 of 30 of the occupied PNG sites were composed of exclusively high-mannose glycans, while the remaining sites contained predominantly processed glycans . The 108MS was used to investigate the disulfide-bond patterns in fractionated gp120 monomers and dimers. The LC-MS data confirmed the canonical disulfide bond between the conserved 18 cysteines in both TV1.C gp120 and 1086.C gp120 monomers  Tables. The antigenic properties of the TV1.C and 1086.C gp120 monomers and dimers were evaluated using surface plasmon resonance (SPR). gp120 monomers from both strains bound the soluble CD4 receptor ligand, CD4-Fc (sCD4), and the anti-V3 mouse mAbs, 1B7A6 and 10B6A8, with affinities in the nanomolar range . OtherwiWe also examined the binding of the TV1.C and 1086.C gp120s to polyclonal sera from 34 HIV+ South African volunteers. Both gp120s bound to all but one of these sera with comparable EC50s ; for SA-The immunogenicity of purified TV1.C and 1086.C gp120s formulated with the MF59 plus carbopol adjuvant , 40, 41 The relative levels of epitope-specific Abs in the sera on immunized rabbits were also investigated. As it might be expected from their similar binding to V3-directed mAbs, the monomeric and dimeric forms from both TV1.C and 1086.C elicited similar levels (~35–40%) of V3-reactive Abs . In contThe results of the RV144 Phase 3 HIV vaccine trial  providedSPR and MS analyses revealed both complementarity and similarities in the antigenicity and glycan profiles of the of TV1.C and 1086.C gp120s. While both gp120s bound sCD4 and two V3-specific mAbs, they showed differential binding to CD4BS mAbs (VRC01 and b12), a mannose-dependent mAb (2G12), V1V2-directed mAbs  and a V2V3 mAb (PG09). Importantly, the presence of specific PNGs and their occupancy differed between the molecules; notably, 1086.C gp120 lacks the N160 and N332 sites recognized by broadly neutralizing Abs (bNAbs) directed to quaternary V1V2 and oligomannose patch epitopes; it should be noted that in this case, although non-occupancy of PNGs may be a direct cause of abolished mAbs binding, lack of quaternary structure can be an equally likely explanation.. HoweverThe glycosylation profiles of these particular gp120’s show a larger number of glycosylation sites occupied exclusively with high mannose glycans compared to many other gp120’s and gp140’s that have been analyzed previously. For example, the trimeric gp140, C.ZA97012 contained only two glycosylation sites that were exclusively occupied by high mannose glycans (37). Similarly, the sequence variant gp140 JRFL contained no sites that contained exclusively high mannose glycans (35). The gp140, B.700010040.C9, which is also a transmitted/founder sequence variant had just six sites occupied exclusively with high mannose glycans (27), compared to TV1.C’s nine sites. The proportion of high mannose glycans for a given gp120 or gp140 is a variable that appears to be sequence dependent. Since it is well known that virion-derived Env has a higher proportion of high mannose glycans, the immunogens studied here, with enhanced high mannose glycans, may perform more favorably than those gp120’s and gp140’s that contain mostly processed glycoforms.More important than measuring the absolute quantity of high mannose glycans in any given immunogen is determining whether or not the high mannose glycans are installed at the correct sites. Recently, a glycopeptide-level analysis of a trimeric-membrane anchored Env (JRFL) was conducted, and it was shown that the increase in high mannose glycans for native trimeric Env only occurs at a few key sites, while other sites retain their processed glycoforms . The keyIn addition, the MS analysis revealed some heterogeneous disulfide bonding, particularly in V1V2-C1, a common feature among recombinant HIV Env antigens (36–39). The dimeric gp120 fraction of TV1.C contained more extensive heterogeneity in its disulfide bonds. Homodimeric peptides, which can result only from intermolecular disulfide bonds, were observed in both C1 and the V1V2 region, suggesting that dimerization occurred in a stochastic manner involving multiple sites.The purified TV1.C and 1086.C gp120 proteins bound well to polyclonal anti-sera from HIV-infected subjects in South African and to well-characterized human mAbs directed to the HIV Env CD4-binding site (CD4BS) and V1V2 domains. Dimeric forms did not bind well to these latter mAbs. When fractionated gp120 monomeric and dimeric forms were used to immunize rabbits using the MF59 adjuvant, they elicited high titer binding and neutralizing Abs against diverse HIV strains with titers highest against Tier 1 pseudoviruses. Of note, the monomeric gp120 preparations induced Abs that recognized CD4BS and V1V2 epitopes while the dimeric forms did not appear to efficiently elicit these specificities.Additional studies utilizing bivalent preparations of the TV1.C and 1086.C gp120s with the MF59 adjuvant as boosts were performed in rhesus macaques comparing priming immunizations with recombinant NYVAC and ALVAC vaccine vectors . FollowiIt is important to note that in a recent re-analysis of the immune correlates of protection risk using a number of V2 antigens, the 1086.C V1V2 protein correlated among the best of all antigens tested . Thus, tIn summary, these results provide support for inclusion of the TV1.C and 1086.C gp120 proteins in future clinical trials. While recent advances in the design of native trimeric HIV Env antigens , 46, 47 S1 Fig(PPT)Click here for additional data file.S2 Fig(PPT)Click here for additional data file.S3 Fig6 cells/ml, left axis) and productivity  of HIV-1 gp120 proteins in the absence of G418.Terminal batch cultures  were initiated from the corresponding parent cultures . Data points indicated by filled circles (●) and open circles (○) represent the amount of viable cell counts (106 cells/ml) and total protein concentration (g/ml), respectively.Cell growth curves Click here for additional data file.S4 Fig(PPT)Click here for additional data file.S5 FigThe y-axis shows Optical Density (OD) read-out at 450nm; the x-axis shows serum (log) concentration. Rabbit # 1 (Ra-1) to Rabbit # 6 (Ra-6) were immunized with TV1.C gp120 monomer; Rabbit # 7 (Ra-7) to Rabbit # 12 (Ra-12) were immunized with TV1.C gp120 dimer; Rabbit # 13 (Ra-13) to Rabbit # 18 (Ra-18) were immunized with 1086.C gp120 monomer; Rabbit # 19 (Ra-19) to Rabbit # 24 (Ra-24) were immunized with 1086.C gp120 dimer. The darker color (filled and open symbols) show binding to respective V3- , V1V2-  and CD4BS-  gp120 mutants. The lighter color (filled and open) symbols indicate control binding. The data points were fitted to non-linear regression  analysis.(PPTX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Tablefive mandatory criteria and three recommended criteria were identified and acceptance criteria set to down-select the top two proteins  for subsequent cell line development and gp120 production to support clinical trial material generation.(PPT)Click here for additional data file.S2 TableThe number of potential glycosylation sites and the number of occupied sites are compared for each potentially glycosylated peptide.(PPT)Click here for additional data file.S3 TableThe number of potential glycosylation sites and the number of occupied sites are compared for each potentially glycosylated peptide.(PPT)Click here for additional data file.S4 Table(PPT)Click here for additional data file.S5 Table(PPT)Click here for additional data file.S6 Table(PPT)Click here for additional data file.S7 Table(PPT)Click here for additional data file.S8 Table(PPT)Click here for additional data file.


Pluripotency transcription programs by core transcription factors (CTFs) might be reset during M/G1 transition to maintain the pluripotency of embryonic stem cells (ESCs). However, little is known about how CTFs are governed during cell cycle progression. Here, we demonstrate that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle genes in determining the identity of ESCs. Aurkb phosphorylates Oct4(S229) during G2/M phase, leading to the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Aurkb phosphor-mimetic and PP1 binding-deficient mutations in Oct4 alter the cell cycle, effect the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Our findings provide evidence that the cell cycle is linked directly to pluripotency programs in ESCs.DOI:http://dx.doi.org/10.7554/eLife.10877.001 Embryonic stem cells can give rise to any type of cell in the body – an ability known as pluripotency. These cells rapidly divide and self-renew until they are exposed to signals that cause them to mature into a particular specialized cell type.As cells prepare to divide, they transition through a series of phases known as the cell cycle. In embryonic stem cells, these phases are often shorter than in other cell types. This altered timing is thought to be important for maintaining the pluripotency of the stem cells.Proteins called core transcription factors also help stem cells to remain pluripotent. Evidence suggests that the activity of some of these proteins affects the timing of the different cell cycle phases. However, it is not clear exactly how they do so or how the activity of the transcription factors is controlled.A core transcription factor called Oct4 is thought to be a “master regulator” of pluripotency that controls the activity of many of the other core transcription factors. Shin, Kim, Kim, Kim et al. have now studied the activity of Oct4 around the point of cell division. This revealed that a protein called aurora kinase B modifies Oct4 by adding a phosphate group to it just before a cell divides. This modification causes Oct4 to detach from chromatin, the protein structure in which DNA is packaged inside cells.Following cell division, another protein called PP1 removes the phosphate group from Oct4. This “resets” the pluripotency of the stem cell, allowing it to continue to self-renew. Cells that contain only mutant forms of Oct4 that cannot bind to aurora kinase B or PP1 lose their pluripotency. The mutant Oct4 proteins also alter the cell cycle of the stem cells.Overall, Shin et al.’s findings suggest that Oct4 regulates the cell cycle of embryonic stem cells as well as their pluripotency. How Oct4 activity affects the specialization of the stem cells into mature cell types remains to be investigated in future studies.DOI:http://dx.doi.org/10.7554/eLife.10877.002 Embryonic stem cells (ESCs) have unique transcriptional programs for self-renewal and pluripotency which differentiates into all types of cells. Core transcription factors—Oct4, Sox2, Nanog (OSN)—govern such pluripotency transcriptional programs . ESCs grOur understanding of the molecular associations between the cell cycle and pluripotency in ESCs is limited. Robust Cdk2 activity shortens G1 phase by inducing rapid G1-S transition and promotes pluripotency and self-renewal in ESCs . Nanog cOct4 is considered a master regulator of ESC pluripotency through its cooperation with other core transcription factors . Post-trThe Aurora kinase b (Aurkb)-protein phosphatase 1 (PP1) axis is critical for kinetochore assembly/disassembly during the cell cycle, regulating the balance between phosphorylation and dephosphorylation of kinetochore substrates . SpecifiIn this study, we demonstrate that the Aurkb-PP1 axis regulates Oct4 during the cell cycle over time and by location. We found that Oct4 contains a well-conserved Aurkb phosphorylation residue (S229) and PP1 binding motif (RVXF) in its homeodomain. Aurkb phosphorylates Oct4(S229) during G2/M phase and dissociates p-Oct4(S229) from chromatin, and PP1 dephosphorylates p-Oct4(S229) during the M/G1 transition, which prompts Oct4 to reset pluripotency transcription on re-entry into the following G1 phase. We found that mutating the Aurkb-phosphorylation residue S229 and the PP1-binding residue F271 of Oct4 in ESCs led to a significant loss of pluripotency and altered the cell cycle. Transduction of these mutants into MEFs significantly decreased the reprogramming efficiency.Based on these findings, we propose that the spatiotemporal regulation of Oct4 by the Aurkb-PP1 axis during the cell cycle is critical for resetting pluripotency and cell cycle genes in determining the identity of ESCs.To understand the function of the phosphorylation of Oct4, we examined its phosphorylation sites by transient transfection of Flag-Oct4 into E14 ESCs, analyzed the phosphorylation state of purified Oct4 by mass spectrometry, and identified 4 phosphorylation sites . We thenNext, we generated a rabbit polyclonal antibody against phosphorylated Oct4(S229) [thereafter p-Oct4(S229)] and confirmed its specificity by dot blot and western blot .We then examined p-Oct4(S229) expression by confocal microscopy in undifferentiated E14 ESCs  levels . In prevTo confirm the localization of p-Oct4(S229), we adapted the fluorescence ubiquitination cell cycle indicator (FUCCI) reporter system to ESCs . We geneWe then confirmed that Oct4 dissociates from the binding region of Oct4 and Nanog at G2/M phase by ChIP-qPCR . Under tTo identify the kinases that phosphorylate Oct4(S229), we selected 19 candidates using a group-based prediction system  and amonAurkb. Further, we confirmed that recombinant Aurkb phosphorylated GST-Oct4(S229) by in vitro 32P-ATP-labeled kinase assay and western blot with anti-p-Oct4(229) (To identify the kinases that mediate Oct4(S229) phosphorylation during G2/M phase, we screened the kinases by administering nocodazole to ESCs that were knocked down with cognate lentivirally expressed shRNAs of kinases . p-Oct4(ct4(229) .To verify the Aurkb-mediated phosphorylation of Oct4(S229), we treated nocodazole-pretreated E14 ESCs (10 hr) with various aurora kinase inhibitors for 15 min. An Aurkb-specific inhibitor, hesperadin, completely blocked the phosphorylation, but an Aurka-specific inhibitor, MLN8237, did not. AT9283, an inhibitor of both Aurka and Aurkb, prevented phosphorylation . Under tWe then observed that Flag-Aurkb interacts with endogenous Oct4 in E14 ESCs by immunoprecipitation . To deteWhen nocodazole treated ZHBTc4 ESCs were released into normal serum, the Aurkb-Oct4 interaction weakened and p-Oct4(S229) levels declined , indicatIn examining the amino acid sequence of Oct4, we found that it contains a protein phosphatase 1 (PP1)-binding sequence (268-RVWF-271) in its homeodomain, near the S229 Aurkb phosphorylation site in the 3-dimensional structure . This moNext, we examined the interaction between Oct4 and PP1 isoforms during the M/G1 transition after nocodazole treatment and release into normal serum . p-Oct46-PP1γ, GST-Oct4(WT), and GST-Oct4(F271A). (His)6-PP1β and γ bound more robustly to GST-Oct4(WT) but weakly to GST-Oct4(F271A) ] by site-directed mutagenesis and measured the in vitro interaction between PP1 and Oct4(WT) or Oct4(F271A) using bacterially purified recombinant (His)4(F271A) , indicatWe then determined whether PP1 dephosphorylates the Aurkb-catalyzed p-Oct4(S229) by preincubating recombinant GST-Oct4 with recombinant Aurkb, adding purified PP1, and measuring the phosphorylation state of p-Oct4(S229) by western blot. Recombinant PP1β and PP1γ, but not PP1α, dephosphorylated Aurkb-mediated phospho-Oct4(S229) . FurtherBased on the findings that p-Oct4(S229) dissociates from condensed chromatin  and PP1 Interestingly, the overall levels of Oct4 protein did not change significantly throughout the cell cycle. However, unlike Oct4, the levels of Nanog, which has a short half-life , rose inTo test that PP1 is required for resetting the transcription of Oct4 target genes, we administered 50 nM OKA to nocodazole-pretreated E14 ESCs for 4 hr and released them into normal serum. As expected, OKA retarded the dephosphorylation of p-Oct4(S229) and re-entry into the next G1 phase .To examine the binding of Oct4 to its targeting pluripotency genes on chromatin during the M/G1 transition, we performed the ChIP-qPCR assay. As a result, Oct4 bound weakly to target genes in G2/M phase, strengthening its association during entry into G1 phase. On treatment with OKA, Oct4 binding to target genes declined significantly during entry into G1 phase . In addiTo analyze Oct4-depenent transcriptional resetting during the M/G1 transition, we measured nascent RNA levels . When E1http://david.abcc.ncifcrf.gov), we identified some Oct4 target genes associated with various cell cycle related functional categories  at G2/M (9204) and G1 (24548) phase  phase . Expecte8) phase . Further8) phase  implicat8) phase . We cate8) phase . For exa8) phase .10.7554/To determine the significance of recycling Oct4 through Aurkb/PP1 in ESC pluripotency, we generated ZHBTc4 ESCs that stably expressing wild-type Oct4(WT), a phosphor-mimic Oct4(S229D) mutant, and a PP1-binding-defective Oct4(F271A) mutant. We confirmed that ectopic wild-type Oct4 and Oct4 mutants were expressed when endogenous Oct4 was removed by doxycycline (dox) treatment .10.7554/Under the same conditions (dox for 2 days), we analyzed the cell cycle patterns in these ESCs . Oct4-deTo determine the reset patterns of Oct4 mutants during the M/G1 transition, we analyzed nascent RNA levels of a subset of Oct4 target genes in ZHBTc4 ESCs harboring Oct4(WT) and Oct4 mutants that were pretreated with dox for 2 days, given nocodazole (6 hr), and released into normal serum .Nascent To determine the long-term effects of Oct4 mutations on ESC pluripotency and cell cycle progression, we cultured ZHBTc4 ESCs containing Mock, Oct4(WT), Oct4(S229D), and Oct4(F271A) for 7 days with dox. Ectopic Oct4(WT) and Oct4 mutants  were continuously expressed until dox treatment for 7 days . Total mTo determine the effects of mutation on Oct4 transcriptional activity, we infected retroviral wild-type and mutant Oct4 into NIH-3T3 cells in which a 10X Oct4 response element (RE)-driven luciferase reporter was stably incorporated and measured luciferase activity. Oct4(F271A) and Oct4(S229D) showed little luciferase activity . Oct4S2. In addiBased on our findings, we propose that the recycling of Oct4 by Aurkb/PP1 over time and by location is pivotal for the transcriptional resetting of Oct4 during entry in to the subsequent G1 phase, ultimately maintaining ESC pluripotency and cell cycle progression .In this study, we have demonstrated that Oct4, a master pluripotency transcription factor, is spatiotemporally regulated by the Aurkb-PP1 axis during the cell cycle. In G2/M phase, Aurkb phosphorylates Oct4 extensively at serine 229, leading to its dissociation from chromatin . The detAurka regulates ESC pluripotency through phosphorylation-mediated inhibition of p53  but is nWe also found that PP1 mediates the resetting of Oct4 during the M/G1 transition. PP1 governs the resetting of cell cycle machinery , bindingIn addition, we revealed that PP1 inhibition by OKA treatment delayed the Oct4 dephosphorylation at S229 residue and consequently resetting to the target genes in E14 ESCs during M/G1 transition . We furtWe confirmed that Aurkb-phosphor-mimetic and PP1-binding-defective mutations lead to a loss of pluripotency, even in the presence of LIF, and that introduction of these mutants into somatic cells lowers the reprogramming efficiency , supportOct4 RNA levels fluctuated during the cell cycle , and 1000 U/ml ESGRO . Aurora kinase inhibitors AT9283, Hesperadin and MLN8237 were purchased from Selleckchem  and okadaic acid from Sigma.E14 and ZHBTc4 ESCs were cultured as described  in 0.1% For G2/M phase synchronization, E14 cells were treated with 200 ng/ml nocodazole  for 10 hr and ZHBTc4 cells were treated with same concentrations of nocodazole for 6 hr. In order to release, synchronized cells were washed three times with PBS, and incubated for the indicated time in fresh culture media.For cell cycle analysis, the collected cells at the indicated time were immediately fixed in 70% ethanol and stained with propidium iodide  for 1 hr at room temperature in the dark. Cell cycles were analyzed using FACSCalibur flow cytometer and LSRII (SORP) . Analysis of cell cycle data was performed with FlowJo .Cells grown on coverslips were fixed in 4% (w/v) paraformaldehyde and permeabilized in 0.5% (w/v) Triton X-100 in PBS for 30 min at room temperature (RT). After permeabilization, the cells were blocked with 3% (w/v) BSA for 30 min. Subsequently, they were incubated in primary antibody for 1 hr at RT. Antibody dilutions were 1:500 for anti-Oct4 , 1:200 for anti-p-Oct4(S229), 1:200 for pH3S10 . Secondary antibodies used in immunostaining were Alexa Fluor 488, 568 .Confocal micro-images were obtained by a confocal laser scanning microscope .The plasmid pGAE-mKO2:Cdt1 and pGAE-mAG:Geminin were generously provided by Savatier, P. . For stable expression in ESCs, fragments containing mKO2:Cdt1 and hmAG1:Geminin coding sequences, respectively, were generated by PCR amplification and were sub-cloned between the SalI and AgeI sites into pCAG-IP vector to generate pCAG-mKO2:Cdt1-IP and pCAG-mAG:Geminin-IP. PP1α, β and γ were amplified by PCR from cDNA of E14 ESCs. The PCR products were digested with XhoI and AgeI then subcloned into pCAG-Flag-IP vector. All Oct4 mutants were generated by site-directed mutagenesis .For long-term transgene expression in ZHBTc4 ESCs, Flag-tagged Oct4 was cloned into pCAG-Flag-IP, which was generated by inserting a Flag tag into pCAG-IP, kindly provided by Hitoshi Niwa . To generate ESCs stably expressing Flag-Oct4, pCAG-Flag-Oct4-IP was transfected into ESCs using Lipofectamine (Invitrogen). After 48 hr of transfection, selection with 2μg/ml of puromycin was performed to determine stable integration. Puromycin resistant cells were expanded and analyzed for Oct4 by Western blot.The reporter gene assay was done as described . Briefly0.2 μg of purified GST-Oct4 proteins were used in cold in vitro kinase assays with purified recombinant kinases. All kinases were purchased from Proqinase (Germany). Kinase reactions were performed in kinase buffer  for 30 min at 30°C. Then reactions were stopped by the addition of 5X SDS-PAGE loading buffer and assessed by Westernblot.32P-ATP  for 30 min at 30°C. Reactions were then stopped by the addition of 5X SDS-PAGE loading buffer and loaded for separation on 8% SDS-PAGE gel. After staining with Coomassie Blue, the gels were dried and exposed to films.For radioactive in vitro kinase assay GST-Oct4 was incubated with Aurkb in kinase buffer  with 0.1 µM γ-In vitro phosphatase assay was performed as described . BrieflyImmunoprecipitation and Western blot were performed as described . Anti-p-To prepare nascent RNA, Click-iT Nascent RNA Capture Kit  was used. First, ESCs were incubated with 100 μM of 5-ethynyl uridine (EU) for 15 min. After incubation, EU-labeled RNA was isolated and converted into biotinylated RNA by Click reaction. The biotinylated RNA was pulled down with streptavidin magnetic beads. cDNA was synthesized with RNA bound to the beads as a template and analyzed by qRT-PCR. The primers that span an intron–exon boundary were used for specifically detecting of nascent RNA. The primer sequences used in the analysis are given in Lentiviruses were produced using PLKO-puro constructs that express shRNAs; Aurka#2(TRCN0000025140), Aurka#5(TRCN00000251430), Aurkb#2(TRCN0000374361), Aurkb#3(TRCN0000321718), Pak1#4(TRCN0000025258), Pak2#1(TRCN0000025209), Pak2#5(TRCN0000413412), Pkca#2(TRCN0000022875) and Pkca#5(TRCN00002187830) were purchased from Sigma. 293FT cells were cotransfected with 0.5 μg each of pMD2.G, pMDLg/pRRE, pRSV-rev, and 1 μg of pLKO-shRNA using Lipofectamine (Invitrogen) in a 6-well plate. 48 hr after transfection, virus-containing medium was collected and passed through 0.45-μm filters. Polybrene (10 μg/ml) was added to target cells immediately prior to infection, and infection was performed for 5 hr. Target cells were selected with puromycin (2 μg/ml) 48 hr after infection.Preparation of RNAs, reverse transcription PCR, real-time qPCR, and chromatin immunoprecipitation (ChIP) assay were done as described . The prihttp://www.lascience.co.kr). For the ChIP-seq analysis, reads were mapped to the mouse genome (NCBI build 37/ mm9). The detailed analysis was done as described (We ChIPed with Oct4 antibody at G2/M (nocodazole-treated cells) and G1 phase (release of nocodazole-treated cells) in E14 ESCs. ChIPed DNAs were sequenced by LAS  was performed as described . ESCs weReprogramming was performed as described . BrieflyNano-LC-ESI-MS/MS Analysis of Phosphorylation Sites in Oct4.Nano-LC-ESI-MS/MS analysis was previously performed as described . 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 submission has been favorably evaluated by Fiona Watt (Senior editor), a Reviewing editor, and three reviewers.Thank you for submitting your work entitled "Aurkb/PP1-Mediated Resetting of Oct4 During the Cell Cycle Determines the Identity of Embryonic Stem Cells" for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.All reviewers found the work to be of considerable interest but wanted additional work to reinforce some of the most critical conclusions. Given that virtually nothing is known about how post-translational modifications regulate Oct4, this paper provides some provocative evidence that phosphorylation by Aurora B Kinase influences the association of Oct4 with chromatin. Addressing the critical points enumerated below will strengthen the paper:1) Please determine when in the cell cycle Oct4 is bound to DNA and perform CHIP-seq during distinct phases of the cell cycle  to define which regions are bound in a cell-cycle dependent manner.2) Please provide evidence for the robustness of the pOct4 Ab for CHIP.3) Consider the criticism from Reviewer #2 regarding the lack of specificity of the OKA experiments.4) Provide better characterization of the nocodozole effects as stressed by Reviewer #3.Reviewer #1:The manuscript describes data showing that Oct-4 is phosphorylated by aurora kinase B in the G2/M phase of the cell cycle of mouse embryonic stem cells (mESCs). This mechanism blocks Oct-4 to bind chromatin. PP1 then dephosphorylate Oct-4 in M/G1 restoring its transcriptional activity. Inhibition of this mechanism inhibits pluripotency and decrease cellular reprogramming efficiency. The authors propose that this mechanism could reset the pluripotent state of mouse ESCs at each cell cycle thereby allowing for a short period of time the cells to differentiate.These data are interesting and novel. They offer new insights concerning the mechanisms by which cell cycle could control cell fate choice in stem cells. Nonetheless few aspects could be clarified.The precise timing by which Oct-4 is on the chromatin is unclear. Indeed, it would be expected that Oct-4 may leave naturally its binding site to allow DNA replication during S phase. So, It would be interesting to check if Oct-4 is binding DNA in S phase? What happening to its phosphorylation state in S phase? How does it relate to the S229 phosphorylation?The authors should cite the recent manuscript published by Huck Hui Ng and colleagues PMID: 26232226. How do their results relate to these mechanisms described in this publication?In addition, the authors only look at few OCT-4 binding site in G2/M. Indeed, their data suggest that Oct-4 can still bind DNA during this phase of the cell cycle. It would be interesting to identify the genomic regions bound by Oct4 in G2/M and M/G1.Reviewer #2: In this study, Shin et al. demonstrated that Oct4, a key regulator of pluripotent stem cells, is phosphorylated at S229 at G2/M phase but dephosphorylated at G1 phase of each cell cycle. The authors convincingly demonstrated that the Aurora kinase B and PP1, which play critical roles in kinetochore assembly and disassembly, regulate this modification. The authors argue that dephosphorylation of S229 at each G1 phase allows resetting the core transcriptional network. Overall, this study discovered a novel and interesting post-translational modification of Oct4, however, the biological consequence/relevance of this regulation is not established.The main concerns are:1) Data demonstrating that p-Oct4 dissociates from chromatin is not convincing. The main evidence supporting this point is 2) Because suppression of PP1 by OKA affects activities of many other PP1 substrates, the observed differences in cell cycle progression, chromatin binding, and nascent mRNA transcription in 3) In 4) It is difficult to fully appreciate the functional relevance of S229 modification. It seems that WT Oct4 is better than both S229A and S229D mutants in self-renewal and/or reprogramming assays . Why phoReviewer #3:In this manuscript, the authors investigated the phosphorylation sites on the core pluripotency factor Oct4 and associated one of the phosphorylation site S229 with Aurora B and PP1 which are key cell cycle kinases and phosphatase. Interestingly, the phosphorylation status on Oct4 S229 seems to modulate Oct4 chromatin binding activity. And the authors proposed that the dynamic phosphorylation of Oct4 is important for its ejection and rebinding before and after mitosis. Although the precise role of Oct4 ejection and rebinding within a cell cycle is not clear, the authors hinted that this could be important to maintaining Oct4 transcription and pluripotency.Overall, this is an interesting manuscript which links cell cycle kinase/phosphatase with the controlling of the activity of the key pluripotency factor Oct4. The authors performed nice biochemical and functional experiment to investigate this novel phosphorylation site on Oct4. Although some of the experiments are more correlative, overall the experiments support their conclusions.Specific comments:1) Does the concentration of nocodazole used in this study arrest cells in G2 phase or M phase? Aurora B is known to be most active in M phase; besides, M phase chromatin condensation are known to cause transcription factors ejection from chromatin. Thus it is better to perform FACS staining for a mitotic marker, so that the authors may be able to precisely locate M phase as the time in the cell cycle when Oct4 gets phosphorylated.2) 3) Aurora B plays an important role in maintaining mitotic arrest caused by spindle disrupting agents, like nocodazole. In 4) If Aurora B kinase is activated exogenously during interphase, will this trigger premature Oct4 phosphorylation and ejection from chromatin during interphase?5) The conclusion on Oct4 phosphorylation and chromatin ejection based on experiments using nocodazole treatment is only correlative. Authors only showed that during mitosis Oct4 gets phosphorylated by Aurora B which correlates with its cytoplasmic localization. However, the majority of transcription factors are ejected from chromatin during mitosis due to chromatin condensation. Thus, the ejection of Oct4 from chromatin during mitosis could be due to the physical condensation of chromatin rather than the phosphorylation by AuroraB.6) Is S229A mutant more potent in binding to Oct4 target genes even with PP1 inhibition? Alternatively, will S229A mutant constitutively bind to chromatin even in M phase? If Oct4 S229A mutant is more potent is chromatin binding, then why does it seem to possess weaker activity and also caused a reduction in reprogramming efficiency? 1) Please determine when in the cell cycle Oct4 is bound to DNA and perform CHIP-seq during distinct phases of the cell cycle  to define which regions are bound in a cell-cycle dependent manner.According to the reviewers’ comments, we mapped the genomewide occupancy of Oct4 at G2/M and G1 phase in E14 ESCs by ChIP-seq. We found high-confidence peaks  at G2/M (9204) and G1 (24548) phase . ExpecteNext to identify reset region by Oct4, we discovered 9092 of genomic regions enriched by Oct4 more than two-fold increase in G1 phase compared to G2/M phase . We cateOn the other hands, we identified G2/M or G1 specific peaks . Majorit2) Please provide evidence for the robustness of the pOct4 Ab for CHIP.The reviewers raise an important point. Throughout the Despite p-Oct4(S229) being pulled down, the chromatin was not. Moreover, it has mentioned that steric and electrostatic clashes induced by phosphorylation at S229 residue can cause loss of DNA binding ability . From above, weak ChIP signal of pOct4(S229) in 3) Consider the criticism from Reviewer #2 regarding the lack of specificity of the OKA experiments.To complement the experiment which may be misrepresented by cell cycle effect of OKA treatment, we performed ChIP-qPCR assay in Zhbtc4 ESCs stably expressing wild-type Oct4 (WT) and phsphor-defect mutant (S229A). When cells were released into G1 phase, OKA treatment significantly prevented the binding of Oct4(WT) to target genes. On the other hand, binding of Oct4(S229A) was relatively less affected even in treatment of OKA .Even though Oct4(S229A) has the ability of binding to target genes, the reason why Oct4(S229A) has the low capacity of reprogramming can be explained by O-GlcNAcylation of Oct4. In our previous study, we identified that serine 228 O-GlcNAcylation of Oct4 is crucial for maintaining pluripotency . We found that Oct4 (S229A) mutation led to reduction of Oct4-O-GlcNAcylation which is critical for Oct4 activity, thereby S229A may reduce DNA binding of Oct4 and reprogramming efficiency. Based on these findings we concluded that dephosphorylation is paramount for the resetting of Oct4 to target genes during the M/G1 transition.4) Provide better characterization of the nocodozole effects as stressed by Reviewer #3.It is reported that during S phase Aurkb is activated then accumulates histone H3 serine 10 phosphorylation (H3S10p), a well-known mitotic marker, in mouse embryonic stem cells . In consistent with this report, when we released G2/M arrested E14 mouse embryonic stem cells, H3S10p levels retained highly through S-G2/M phase (7-10 hrs after release) , indicatReviewer #1:The precise timing by which Oct-4 is on the chromatin is unclear. Indeed, it would be expected that Oct-4 may leave naturally its binding site to allow DNA replication during S phase. So, It would be interesting to check if Oct-4 is binding DNA in S phase? What happening to its phosphorylation state in S phase? How does it relate to the S229 phosphorylation?We previously showed that p-Oct4(S229) begin to be detected in the S phase by western blot and FACS analysis . AccordiThe authors should cite the recent manuscript published by Huck Hui Ng and colleagues PMID: 26232226. How do their results relate to these mechanisms described in this publication?We thank the reviewer for this suggestion. The study revealed that S and G2 phases are gatekeepers of differentiation in human embryonic stem cells. We cited the paper in the Introduction and Discussion section.In addition, the authors only look at few OCT-4 binding site in G2/M. Indeed, their data suggest that Oct-4 can still bind DNA during this phase of the cell cycle. It would be interesting to identify the genomic regions bound by Oct4 in G2/M and M/G1.According to the reviewer’s comments, we mapped the genomewide occupancy of Oct4 at G2/M and G1 phase in E14 ESCs by ChIP-seq. We found high-confidence peaks . at G2/M (9204) and G1 (24548) phase . ExpecteNext to identify reset region by Oct4, we discovered 9092 of genomic regions enriched by Oct4 more than two-fold increase in G1 phase compared to G2/M phase . We cateOn the other hands, we identified G2/M or G1 specific peaks . MajoritReviewer #2:1) Data demonstrating that p-Oct4 dissociates from chromatin is not convincing. The main evidence supporting this point is Throughout Despite p-Oct4(S229) protein being pulled down, the chromatin was not. Moreover, it has mentioned that steric and electrostatic clashes induced by phosphorylation at S229 residue can cause loss of DNA binding ability . From above, weak ChIP signal of pOct4(S229) in 2) Because suppression of PP1 by OKA affects activities of many other PP1 substrates, the observed differences in cell cycle progression, chromatin binding, and nascent mRNA transcription in At the reviewer’s suggestion, we performed ChIP-qPCR assay in Zhbtc4 ESCs stably expressing wild-type Oct4(WT) and phsphor-defect mutant(S229A). When cells were released into G1 phase, OKA treatment significantly prevented the binding of Oct4(WT) to target genes. On the other hand, binding of Oct4(S229A) was relatively less affected even in treatment of OKA .Even though Oct4(S229A) has the ability of binding to target genes, the reason why Oct4(S229A) has the low capacity of reprogramming can be explained by O-GlcNAcylation of Oct4. In our previous study, we identified that serine 228 O-GlcNAcylation of Oct4 is crucial for maintaining pluripotency . We found that Oct4 (S229A) mutation led to reduction of Oct4-O-GlcNAcylation which is critical for Oct4 activity, thereby S229A may reduce DNA binding of Oct4 and reprogramming efficiency. Based on these findings we concluded that dephosphorylation is paramount for the resetting of Oct4 to target genes during the M/G1 transition.3) In As the reviewer commented, F271A mutation disrupts pluripotency and cell cycle of mouse embryonic stem cells more severely than S229D . Even th… which is unexpected according to the proposed model. Does the S229D mutant retain some level of transactivation activity?We have already published that S229D mutant has lower activity (around 25% ) in comparison with the activity of Oct4 (WT) .Is the F271A mutant dominant negative?In 4) It is difficult to fully appreciate the functional relevance of S229 modification. It seems that WT Oct4 is better than both S229A and S229D mutants in self-renewal and/or reprogramming assays . Why phoWe believe that the cycle of phosphorylation and dephosphorylation of Oct4 during cell cycle is important. Throughout this study, we revealed that the resetting of Oct4 occurs rapidly during the exit from mitosis to prevent postmitotic cell fate changes in ESCs. In addition, we suggested that the potential of ESCs to differentiate might be derived from the small window of the M/G1 transition and phosphorylation of Oct4 is deeply involved in this mechanism.Is S229D more stable than S229A in mitosis?We treated cycloheximide to Zhbtc4 embryonic stem cells stably expressing each Oct4 WT and mutant form and assessed by western blot . We coulIs the S229A as good as WT Oct4 in activating target genes and supporting self-renewal?To address this question, we performed ChIP-qPCR assay in Zhbtc4 ESCs stably expressing wild-type Oct4(WT) and phsphor-defect mutant(S229A). When cells were released into G1 phase, OKA treatment significantly prevented the binding of Oct4(WT) to target genes. On the other hand, binding of Oct4(S229A) was relatively less affected even in treatment of OKA . Even thAre great majority or only a small fraction of Oct4 molecules phosphorylated by Aurkb at G2/M?At this point, we showed that large portion of phosphorylated Oct4 (S229) was detected in cytoplasm at G2/M phase .Reviewer #3:1) Does the concentration of nocodazole used in this study arrest cells in G2 phase or M phase? Aurora B is known to be most active in M phase; besides, M phase chromatin condensation are known to cause transcription factors ejection from chromatin. Thus it is better to perform FACS staining for a mitotic marker, so that the authors may be able to precisely locate M phase as the time in the cell cycle when Oct4 gets phosphorylated.It is reported that during S phase Aurkb is activated then accumulates histone H3 serine 10 phosphorylation (H3S10p), a well-known mitotic marker, in mouse embryonic stem cells . In consistent with this report, when we released G2/M arrested E14 mouse embryonic stem cells, H3S10p levels retained highly through S-G2/M phase (7-10 hrs after release) , indicat2) To address this question, we mapped the genomewide occupancy of Oct4 at G2/M (nocodazole-treated cells) and G1 phase (release of nocodazole-treated cells) in E14 ESCs by ChIP-seq. We found that Oct4 bound reduced regions of genome at G2/M phase, compared to G1 phase . Further3) Aurora B plays an important role in maintaining mitotic arrest caused by spindle disrupting agents, like nocodazole. In The reviewer raises an important point. Indeed, inhibition of cell cycle related proteins such as Aurkb can result in cell cycle arrest. Consequently, we analyzed cell cycle of E14 mouse embryonic stem cells treated with Aurkb inhibitor under the same conditions applied in Similarly, the use of OKA seems to enrich cells towards 4N, will this indirectly promote Oct4 phosphorylation?When G2/M arrested cells were released into normal serum, OKA treatment retarded re-entry into the next G1 phase . In spit4) If Aurora B kinase is activated exogenously during interphase, will this trigger premature Oct4 phosphorylation and ejection from chromatin during interphase?It has been reported that Aurkb is tightly regulated in a cell cycle dependent manner . Usually, Aurkb in cancer cells is activated during G2/M phase by INCENP-hespin kinase. In addition, in mouse embryonic stem cells, Aurkb is reported to be active during S-G2/M phase . We also showed that Aurkb begin to phosphorylate Oct4 at S phase . In addi5) The conclusion on Oct4 phosphorylation and chromatin ejection based on experiments using nocodazole treatment is only correlative. Authors only showed that during mitosis Oct4 gets phosphorylated by Aurora B which correlates with its cytoplasmic localization. However, the majority of transcription factors are ejected from chromatin during mitosis due to chromatin condensation. Thus, the ejection of Oct4 from chromatin during mitosis could be due to the physical condensation of chromatin rather than the phosphorylation by AuroraB.We thank the reviewer for pointing out this concern. We found several lines of evidence implying that dissociation of phosphorylated Oct4 from chromatin proceeds to mitotic chromatin condensation and explained detailed descriptions in response to question #1. We agree with the reviewer’s opinion that M phase chromatin condensation partly contributes the ejection of Oct4 from chromatin.6) Is S229A mutant more potent in binding to Oct4 target genes even with PP1 inhibition? Alternatively, will S229A mutant constitutively bind to chromatin even in M phase? If Oct4 S229A mutant is more potent is chromatin binding, then why does it seem to possess weaker activity and also caused a reduction in reprogramming efficiency?According to the reviewer’s comment’s, we compared DNA binding ability of Oct4(S229A) to Oct4(WT). Oct4(S229A) binding is slightly higher than Oct4(WT) at G2/M phase and G1 phase with OKA treatment. While, Oct4(S229A) have lower reporter activity in NIH-3t3 cells  and reprReferencesCarmena, M., Wheelock, M., Funabiki, H., and Earnshaw, W.C. (2012). The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. Nature reviews. Molecular Cell Biology 13, 789-803. doi:10.1038/nrm3474Jang, H., Kim, T.W., Yoon, S., Choi, S.Y., Kang, T.W., Kim, S.Y., Kwon, Y.W., Cho, E.J., and Youn, H.D. (2012). O-GlcNAc regulates pluripotency and reprogramming by directly acting on core components of the pluripotency network. Cell Stem Cell 11, 62-74. doi:10.1016/j.stem.2012.03.001Mallm, J.P., and Rippe, K. (2015). Aurora Kinase B Regulates Telomerase Activity via a Centromeric RNA in Stem Cells. Cell Rep 11, 1667-1678. doi:10.1016/j.celrep.2015.05.015Saxe, J.P., Tomilin, A., Scholer, H.R., Plath, K., and Huang, J. (2009). Post-translational regulation of Oct4 transcriptional activity. PLOS ONE 4, e4467. doi:10.1371/journal.pone.0004467


We found that mice immunized subcutaneously with Bac-HAm constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions  in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HA Prior to 2003, H7 subtype avian influenza virus causing human infection cases were very rare and mostly caused by occupational accidents or laboratory exposures –3. For tIn our previous vaccine study, mice that were intranasally immunized with live Bac-HA were protected from lethal H7N7 viral challenge. However, no protection was observed when Bac-HA or inactivated H7N7 virus were administered subcutaneously, possibly due to diminished immunogenic nature of the H7N7 (H7N7/NL/219/03) virus –11. The Interestingly, in our recent vaccine study, mice that were subcutaneously immunized with live Bac-HA (H7N9) survived in both H7N9 and H7N7 virus challenge . Compari2 inhalation for five minutes.All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Temasek Life Sciences Laboratory, National University of Singapore, Singapore. . All animal experiments were carried out in animal BSL3 containment facility in compliance with CDC/NIH and WHO recommendations. Mice were housed in individually ventilated cages  provisioned with water and standard food, and monitored daily for health condition. More than 25% body weight loss was used as criterion for early euthanasia. The animals were euthanized by CO50) of reassortant viruses were calculated by the Muench-Reed method (1938) [Reassortant influenza virus RG-H7N7 (A/Netherland/219/03) and RG-H7N9 (A/Shanghai/2/2013) were generated by reverse genetics as described previously . Virusesd (1938) . All expd (1938) , 28.Spodoptera frugiperda (Sf9II) cells (ATCC) were maintained in Sf900II serum free medium  at 28°C for wild-type baculovirus (wt-Bac) and recombinant baculovirus production.m constructs preparation. For preparing the mutant constructs , we substituted the amino acid alanine at 143, alanine at 198 and valine at 211 coding gene inH7N7HA wild-type cDNA by using quick change site directed mutagenesis kit (Agilent Technologies). Transfection and viral amplification were carried out according to the Baculovirus Expression System Manual (Invitrogen Life technologies).The live baculovirus content of each vaccine constructs were determined by TCID50 method as described Reed and Munch (1938)[The full length of H7N7HA (A/NL/219/03) cDNA gene was commercially synthesized based on the sequences from the NCBI influenza database . cDNA was used for Bac-HA and Bac-HAch (1938). HA contch (1938).m constructs or wt-Bac at 0.5 MOI. Two days post infection, the cells were fixed with 4% formaldehyde and stained with mouse anti H7N7-HA monoclonal antibody (8H9), followed by FITC conjugated goat anti mouse immunoglobulin . The fluorescence signal was detected with an inverted fluorescence microscope  and the images were captured by a digital imaging system .The Sf9 cells were infected with Bac-HA or Bac-HAAmino acid substitution at T143A in H7N7HA wild-type protein inhibits synthesis of N- glycosylation motif in A141. It was confirmed by western blot analysis. Briefly, the Bac-HA or Bac-HA mutant infected cell supernatant was mixed with Laemmli sample buffer and resolved in 12% SDS–PAGE. The gel was transferred to a nitrocellulose membrane and Western blotting was performed as described previously Kumar et al (2013).The antim constructs  containing 2.5μg of HA content (approximately1 X107to 1X107.5 pfu/ml recombinant Baculovirus—Live baculovirus content of each recombinant constructs were determined by using TCID50 method as described Reed and Munch (1938) [8 PFU) and PBS control. Four weeks after the first immunization, a second dose of vaccine was given to all immunized mice groups. Two weeks after the second immunization (42ndday), serum samples were collected from each experimental mice group (Five mice/Group). All samples were stored at -20°C for measuring the hemagglutination inhibition (HAI) and microneutralization (MN) assay against RG-H7N7, RG-H7N9 viruses.Six to eight weeks old specific pathogen free female BALB/c mice were used in all experiments. Mice (10 mice per group/9 groups) were subcutaneously immunized with 100 μL of purified live Bac-HA or Bac-HAh (1938) ) or wt-B50 of RG-H7N7 or RG-H7N9 viruses and incubated at room temperature for 1 h. The mixtures were added to the MDCK monolayers in triplicate wells. One hour after infection, the virus-serum mixture was removed, and serum free DMEM was added to each well. Cytopathic effect was observed 3 to 4 days after incubation at 37°C. The highest serum dilutions that completely protected cells from cytopathology were considered to be the MN titer.The MN assay was performed as described previously . BrieflyVibrio cholera filtrate  for 18 h at 37°C. After addition of 3 volumes of 2.5% (v/v) sodium citrate, the serum samples were incubated at 56°C for 30 min and diluted with PBS to yield a 1:10 dilution of the original serum sample. Serum samples were 2-fold serially diluted in PBS (25 μL sample volume) in Nunc 96-well polystyrene V-bottom microwell plates . Approximately four HA units of each H7 subtype (RG-H7N7 or RG-H7N9) viral antigen was incubated with the serum for 30 min at room temperature followed by the addition of 1% chicken RBCs and incubation at room temperature for 40 min. The inhibition of hemagglutination at the highest serum dilution was considered the HAI titer of the serum.Functional serum antibody titers were determined by hemagglutination inhibition assay .Serum sa50 of mouse adapted RG-H7N7 virus. Mice were observed daily to monitor body weight, clinical signs of disease  and mortality. Mice were humanely euthanized with CO2 inhalation if their body weight dropped to 75% of baseline weights. For determination of lung viral titers after challenge, three mice from each vaccinated group was euthanized by CO2 inhalation on day 5 post-challenge as described Prabakaran et al. [To assess the protective efficacy of the vaccines, immunized mice groups were anesthetized intraperitoneally with ketamine (100 mg/kg)/Xylazine (20 mg/kg) and intranasally challenged with 50 μL (25 μL per naris) of 5 MLDn et al. .The immune epitope Database (IEDB) analysis resources were used  for predP<0.001.All data are expressed as arithmetic mean ± standard deviation. The significance between the groups was calculated using unpaired two-tailed Student’s t test and significance was expressed as ** m protein expressions were analyzed by immunofluorescence staining and their native structure were analyzed by hemagglutination activity with Chicken RBCs. Fluorescence signal and agglutination activity (32 HA units) were detected only in baculovirus expressed H7N7HA (Bac-HA or Bac-HAm constructs) infected cells. In contrast, no fluorescence signal and agglutination activity was observed in cells infected with wt-Bac  assay . Sera ofe groups .50 of homologous H7N7 reassortant virus. The sera obtained from the s.c. vaccinated Bac-HAm construct T143A (1:208), T198A-I211V and I211V-T143A showed a higher neutralizing titer (1:256) against RG-H7N7virus compared to other vaccinated groups. Also, we tested cross-neutralization activity of vaccinated sera against RG-H7N9 virus (m construct T143A(1:128) or T198A-I211V (1:112) vaccinated group showed a higher neutralization activity against H7N9 compared to other vaccinated groups but no significant difference compared to mice immunized with T143A-I211V. Bac-HA vaccinated mice group showed significantly low level of neutralization activity to H7N7 (1:28) or H7N9 (1:10) RG viruses compared toT143A or T198A-I211V or T143A-I211V Bac-HAm construct vaccinated group , I211V (83.33%), and T143A-T198A (50%) also give partial protection against lethal H7N7 challenging, but Bac-HA administered by s.c. route, failed to protect the mice  had significantly higher neutralizing activity against homologous RG-H7N7 virus when compared to the sera from mice vaccinated with Bac-HA and other Bac-HAm. This higher neutralizing response against H7N7 virus could be attributed to the amino acid substitution at position 143, which inhibits the N-linked glycosylation with Asn141 in mutant HA (143A) protein. The lack of glycosylated motif may, in turn, expose the hidden neutralizing epitope at region 138–150  induces higher HAI and neutralization titer is yet to be known and we hope that the mechanism could be elucidated in our future studies.This higher HAI titer was also true when we tested the neutralizing activity of sera obtained from mice immunized with Bac-HA 138–150 , thus, i 138–150 , therebym constructs T143A, T198A-I211V and T143A-T211A showed notably higher neutralizing activity against RG-H7N9 virus than those sera drawn from mice vaccinated using Bac-HA and other Bac-HAm. Based on the epitope prediction result (m (T143A) protein, similar to those found in H7N9 virus, results in the exposure of the B-cell epitope, between region 138 and 150. This explains why antiserum of mice vaccinated with H7N7m constructs has higher cross neutralizing activity against H7N9 virus.A significant association between complete protection against RG-H7N7 influenza virus and titers of HI and neutralization antibody was observed. Previous study has also shown that higher neutralizing antibody and HI titer does protect mice from H7N9 , hence, Based on T-cell epitope prediction, H7N7 and H7N9 viruses, have a T-cell epitope at position 203–211 of their HA proteins. One difference between the amino acid sequence of H7N9 and H7N7 T-cell epitope is at position 211. Several previous studies have suggested that T-cell epitope containing amino acid valine at position 9 acts as a dominant C-terminal anchor residue that provides higher binding affinity to MHC, hence inducing greater antigen-specific T cell immune responses –42. Simim constructs T143A, I211V-T143V and T198A-I211V completely protects the mice that were challenged with 5MLD50 of RG-H7N7 influenza virus. The complete protection observed could be the effect of higher HAI titer and neutralization antibody titer against H7N7 influenza virus. In addition to the 3 constructs just mentioned, vaccinations using 2 other Bac-HAm constructs T198A and T143A-T198A have provided 33.3% and 50% protection respectively. Moreover, immunization of mice using mutant (I211V) HA construct provides sufficient protection (83.33%) when challenged with RG-H7N7. Such reasonable protection could be the effects of higher T-cell responses against H7N7HA. In contrast, no protection was observed in mice vaccinated with Bac-HA, likely a result of the significantly lower level of HAI titer induced by subcutaneous vaccination with Bac-HA construct. Furthermore, the neutralizing titer of sera, from mice vaccinated with Bac-HAm constructs T143A, T198A-I211V and T143A-I211V were significantly higher than that of Bac-HA vaccinated mice  or subunit vaccines or viral like particle vaccines by identifying and inhibiting the potential glycan motif-epitope masks formation in HA protein by amino acids substitution. Even though we could not provide more and precise mechanism regarding how I211V mutation enhancing the immunogenicity, the animal protection against lethal challenge with H7N7 virus data suggest that we can improve antigen specific T cell efficacy by amino acids substitution at T cell epitopes, which provide higher binding affinity to MHC and induce higher antigen specific T cell response.


Human immunodeficiency virus type (HIV)-1 circulating recombinant form 08_BC (CRF08_BC), carrying recombinant reverse transcriptase (RT) gene from subtype B and C, has recently become highly prevalent in Southern China. As the number of patients infected by CRF08_BC increases, it is important to characterize the drug resistance mutations of CRF08_BC, especially against widely used antiretrovirals. In this study, clinically isolated virus was propagated in human peripheral blood mononuclear cells (PBMCs) with increasing concentrations of nevirapine (NVP), efavirenz (EFV) or lamivudine (3TC). Three different resistance patterns led by initial mutations of Y181C, E138G and Y188C were detected after in vitro selection with NVP. Virus variants with initial mutations, in combination with three other previously reported substitutions  or novel mutations  developed during NVP selection. EFV-associated variations contained two initial mutations (L100I and Y188C) and three other mutations . Phenotypic analyses showed that E138R, Y181C and G190A contributed high level resistance to NVP, while L100I and V106L significantly reduce virus susceptibility to EFV. Y188C resulted in a 20-fold reduction of susceptibility to both NVP and EFV. M184V was selected by 3TC as expected. This mutation, alone or with V90I or D67N, decreased 3TC susceptibility by over 1000 folds. These results have brought new insight into the development of drug-related mutations in patients and provided useful information for the optimization of antiretroviral regimens.


TCF7L2, HMGA2 and KCNQ1) and two novel loci (HLA-B and INS-IGF2) at genome-wide significance  = 1.09 to 1.36). Fine-mapping revealed that 88 of 158 previously identified T2D or glucose homeostasis loci demonstrated nominal to highly significant association (2.2×10−23 < locus-wide P<0.05). These novel and previously identified loci yielded a sibling relative risk of 1.19, explaining 17.5% of the phenotypic variance of T2D on the liability scale in African Americans. Overall, this study identified two novel susceptibility loci for T2D in African Americans. A substantial number of previously reported loci are transferable to African Americans after accounting for linkage disequilibrium, enabling fine mapping of causal variants in trans-ethnic meta-analysis studies.Type 2 diabetes (T2D) is more prevalent in African Americans than in Europeans. However, little is known about the genetic risk in African Americans despite the recent identification of more than 70 T2D loci primarily by genome-wide association studies (GWAS) in individuals of European ancestry. In order to investigate the genetic architecture of T2D in African Americans, the MEta-analysis of type 2 DIabetes in African Americans (MEDIA) Consortium examined 17 GWAS on T2D comprising 8,284 cases and 15,543 controls in African Americans in stage 1 analysis. Single nucleotide polymorphisms (SNPs) association analysis was conducted in each study under the additive model after adjustment for age, sex, study site, and principal components. Meta-analysis of approximately 2.6 million genotyped and imputed SNPs in all studies was conducted using an inverse variance-weighted fixed effect model. Replications were performed to follow up 21 loci in up to 6,061 cases and 5,483 controls in African Americans, and 8,130 cases and 38,987 controls of European ancestry. We identified three known loci  in African Americans than in Europeans, recent genome-wide association studies (GWAS) were examined primarily in individuals of European ancestry. In this study, we performed meta-analysis of 17 GWAS in 8,284 cases and 15,543 controls to explore the genetic architecture of T2D in African Americans. Following replication in additional 6,061 cases and 5,483 controls in African Americans, and 8,130 cases and 38,987 controls of European ancestry, we identified two novel and three previous reported T2D loci reaching genome-wide significance. We also examined 158 loci previously reported to be associated with T2D or regulating glucose homeostasis. While 56% of these loci were shared between African Americans and the other populations, the strongest associations in African Americans are often found in nearby single nucleotide polymorphisms (SNPs) instead of the original SNPs reported in other populations due to differential genetic architecture across populations. Our results highlight the importance of performing genetic studies in non-European populations to fine map the causal genetic variants. P<1×10−5) and two candidate SNPs near the p value threshold  totaling 51 SNPs in 21 loci were followed up for replication. rs231356 is 14 kb downstream of the reported T2D index SNP, rs231362, in Europeans HLA region in Europeans in silico and de novo replication in up to 11,544 African American T2D cases and controls, as well as in silico replication in 47,117 individuals of European ancestry from DIAGRAMv2 n≤35,371, n≤82,488, From stage 1 meta-analysis, 49 SNPs moderately associated with T2D (P<5×10−8). Stage 1 meta-analysis identified the established TCF7L2 locus. Stage 1+2a meta-analysis identified the established KCNQ1 and HMGA2 loci. Stage 1+2a+2b meta-analysis identified a second signal at KCNQ1 and a novel HLA-B locus. Secondary analysis including body mass index (BMI) adjustment in stage 1+2a meta-analysis identified the second novel locus at INS-IGF2 (Phet>0.001) (Five independent loci reached genome-wide significance (INS-IGF2 . None oft>0.001) .TCF7L2 locus, the most strongly associated SNP in stage 1+2a African Americans samples was rs7903146  P = 3.4×10−19) P = 2.44×10−15) At the KCNQ1 . Using data from individuals of African ancestry in Southwest USA (ASW) from the 1000 Genomes Project (1KGP) 2 = 0 with rs2283228 in both ASW and CEU) , located 144 kb upstream of the first signal. rs231356 is located at the same LD interval as the index SNPs rs231362 in Europeans Two association signals were observed at KCNQ1 . The firHMGA2 locus, the most strongly associated SNP was rs343092  (HLA-B encodes the class I major histocompatibility complex involved in antigen presentation in immune responses.Two novel T2D loci were identified. The effect sizes of rs2244020 located near 57×10−9) . HLA-B eINS-IGF2 was rs3842770 in African Americans . Most of the 17 T2D-associated SNPs that showed consistent direction of effects had similar effect sizes between this study and prior reports, despite that rs10440833 at CDKAL1 had substantially stronger effect size in Europeans (OR = 1.25) than in African Americans . Additionally, 3 out of 54 trait-increasing alleles from glucose homeostasis-associated index SNPs were associated with increased T2D risk in African Americans (P<0.05).We investigated index SNPs from 158 independent loci associated with T2D and/or glucose homeostasis from prior genome-wide and candidate gene studies in individuals of European, East Asian, South Asian, or African American ancestry . Among t2≥0.3 with the previously reported index SNPs and results were corrected for the effective number of SNPs locusP<0.05, corrected for LD in ASW for SNPs within a locus; P = 0.255, Wilcoxon rank sum test; and P = 0.295, Wilcoxon signed-rank test, respectively, vs. 1.05). The distributions and pairwise differences of risk allele odds ratios were significantly different  . Neither the reported index SNP rs896854 nor its proxies from the CEU data demonstrated significant association to T2D in African Americans . Given rs231362 was modestly associated with T2D in African American (P = 0.04) and was in weak LD (r2 = 0.21 to 0.46 in CEU) with other associated SNPs in this region . Despite rs1531343 and its proxies in high LD were not associated with T2D in African Americans (P>0.05), several SNPs in moderate LD, including rs343092, showed nominal to strong associations  . Howevermericans ,b, sugges region ,d, the rciations ,f. TransP values). Of particular note, FTO is suggested to influence T2D primarily through modulation of adiposity in Europeans P = 0.06 and 0.23, respectively)  . The freP>0.001, TCF7L2, KCNQ1, and HMGA2 were located at protein binding sites or were predicted to alter motif affinity for transcription factors implicated in energy homeostasis  We have performed the largest genetic association analysis to date for T2D in African Americans. Our data support the hypothesis that risk for T2D is partly attributable to a large number of common variants with small effects HLA-B and INS-IGF2, have been implicated in type 1 diabetes (T1D) risk in Europeans HLA class II  than HLA class I regions in Europeans http://www.t1dbase.org). In African Americans, T1D individuals showed both shared and unique risk and protective HLA class II haplotypes as compared to European T1D individuals HLA class II (P<1×10−25) than class I regions (P<1×10−5) HLA class I than class II regions in T2D individuals  since there is long range LD in this region. Recently, rs3130501 near POU5F1 and TCF19 was reported for association with T2D in a trans-ancestry meta-analysis 2 = 0.05) and ASW  from 1KGP nor strongly associated with T2D in the stage 1 meta-analysis (P = 0.04). Other potential non-HLA candidate genes may include TNFA which regulates immune and inflammatory response. It has been hypothesized that activated innate and adaptive immune cells stimulate release of cytokines such as TNFα and IL-1β, which promote both systemic insulin resistance and β-cell damage HLA-DQ/DR, GLIS3 and INS in the susceptibility of latent autoimmune diabetes in adults (LADA) and/or T2D PPARG and TCF7L2 was associated with T1D The two novel T2D loci, s HLA-B, . The obsOur results have several implications regarding the genetic architecture of T2D. First, fine-mapping suggests that currently known loci explain more of the risk than previously estimated. Second, the loci conferring the largest risk for T2D appear to act through regulatory rather than protein-coding changes. Third, many, but not all, of the previously identified T2D loci are shared across ancestries. The differential LD structure of African-ancestry populations at shared loci provides an opportunity for fine mapping in trans-ethnic meta-analysis. Fourth, the ∼2.6M MEDIA SNPs achieved only 43.3% coverage of the 1KGP ASW common SNPs, suggesting that risk loci that are specific to African-ancestry individuals are difficult to discover with the genotyping arrays being used. Large-scale sequencing studies, such as those focusing on whole genomes, exomes, and targeted resequencing for associated non-coding regions, will be necessary to further delineate the causal variants for T2D risk in African Americans.in silico replication of GWAS data from eMERGE and IPM Biobank and de novo genotyping in IRAS, IRASFS, SCCS, and WFSM. In general, T2D cases were defined as having at least one of the following: fasting plasma glucose ≥126 mg/dl, 2 hour glucose during oral glucose tolerance test (OGTT) ≥200 mg/dl, random glucose ≥200 mg/dl, oral hypoglycemic agent or insulin treatment, or physician-diagnosed diabetes. All cases were diagnosed at ≥25 years (or age at study ≥25 years if age at diagnosis was not available). For cohort studies, individuals who met the criteria at any of the visits were defined as cases. Controls with normal glucose tolerance (NGT) were defined by satisfying all the following criteria: fasting plasma glucose <100 mg/dl, 2 hour OGTT<140 mg/dl (if available), no treatment of diabetes, and age ≥25 years. For cohort studies, individuals who met the criteria at all visits were defined as controls. All study participants provided written informed consent, except for eMERGE that use an opt out program, and approval was obtained from the institutional review board (IRB) from the respective local institutions. Detailed descriptions of the participating studies are provided in Stage 1 discovery samples included 17 T2D GWAS studies  with up to 23,827 African American subjects . Stage 2 replication samples included up to 11,544 African American subjects , using −4, or imputation quality score <0.5 . Sample and SNP quality controls were performed as with GWAS data.For stage 1 and 2 GWAS studies, genotyping was performed with Affymetrix or Illumina genome-wide SNP arrays. Imputation of missing genotypes was performed using MACH ore <0.5 . For de P<1×10−5 in stage 1 meta-analysis and index SNPs previously reported to be associated with T2D or glucose homeostasis traits. BMI adjustment allows increasing power to detect T2D loci independent of BMI effect and diminish associations at T2D loci with effects modulated through BMI. Logistic regression was used for samples of unrelated individuals. Generalized estimating equations Single SNP association was performed for each study by regressing T2D case/control status on genotypes. To account for uncertainty of genotype calls during imputation, genotype probabilities or dosage were used for association tests in imputed SNPs. The association tests assumed an additive genetic model and adjusted for age, sex, study centers, and principal components. Principal components were included to control for confounding effects of admixture proportion and population structure. Secondary analysis with additional adjustment for BMI was performed for SNPs with P<5×10−8 from the meta-analysis result of all stages, which has better power than the replication-based strategy In stage 1, association results were combined by a fixed effect model with inverse variance weighted method using the METAL software Phet<0.001), meta-analysis results including studies of all stages assessed by the random effect model implemented in GWAMA Among the 51 SNPs carried forward for replication, heterogeneity of effect sizes across studies within each stage was assessed using Cochran's Q statistic implemented in METAL. Meta-analysis results from stages 1 and 2a, stage 1+2a and 2b were used to assess heterogeneity of effect sizes between discovery and replication stages in African Americans, and between African Americans and Europeans, respectively. For SNPs with significant heterogeneous effect size after multiple comparison corrections  using the 1KGP CEU data with r2≥0.3 with the index SNP. All MEDIA SNPs within these bounds were examined for association analysis. All pairwise LD values within each locus were estimated using the 1KGP CEU and ASW data. To estimate the effective number of SNPs at a locus, we retrieved genotypes from the 1KGP ASW data for markers present in MEDIA, estimated the sample covariance matrix from those genotypes, and spectrally decomposed the covariance matrix kλ is the kth eigenvalue of the K×K covariance matrix for the K SNPs in the locus Index SNPs associated with T2D or glucose homeostasis traits from prior GWAS and candidate gene studies were examined for association with T2D in African Americans . For theip and iψ be the risk allele frequency and the corresponding odds ratio at the ith SNP, respectively. Assuming the additive genetic model and independence between SNPs, the contribution to the sibling relative risk sλ for a set of N SNPs is given by K be the disease prevalence. The liability-scale variance N SNPs is given by z representing the standard normal density at TFor each independent locus, we estimated the sibling relative risk using the most strongly associated SNP within that locus. Let 2 = 0.8 threshold. We used all SNPs with minor allele frequencies ≥1% in both MEDIA and the 1KGP ASW sequence data. Coverage was estimated using non-overlapping bins of 1,000 SNPs.The coverage of MEDIA SNPs to the human genome was estimated using HaploView P<0.05 and P<5×10−8, respectively, in stage 1 samples under an additive model. The observed odds ratios among our stage 1 most significantly associated SNPs with P<1×10−5 ranged from 1.11 to 1.56  includes lymphoblastoid cell lines (LCLs), skin, and adipose tissue derived simultaneously from a subset of well-phenotyped healthy female twins from the TwinsUK adult registry 2-transformed expression signals were normalized separately per tissue as follows: quantile normalization was performed across technical replicates of each individual followed by quantile normalization across all individuals. Genotyping was performed with a combination of Illumina arrays . Untyped HapMap2 SNPs were imputed using the IMPUTE2 software package. In total, 776 adipose and 777 LCL samples had both expression profiles and imputed genotypes. Association between all SNPs  within a gene or within 1 Mb of the gene transcription start or end site and normalized expression values were performed with the GenABEL/ProbABEL packages The MuTHER resource  within 1 Mb of the associated SNPs with cis-expression quantitative trait loci (eQTLs) in peripheral blood leukocytes (LCL) and adipose tissue  using HaploReg We examined putative function of non-coding genome-wide significant SNPs and their proxies within 1 Mb  and meta-analysis results (red diamond and line). At (PDF)Click here for additional data file.Figure S2(A) Distributions of risk allele frequencies for the previously reported index SNPs (in black) vs. the MEDIA most strongly associated SNPs (in red) in African Americans from stage 1 meta-analysis. (B) Distributions of odds ratios for risk alleles of the index SNPs (in black) vs. the most strongly associated MEDIA SNPs (in red) in African Americans from stage 1 meta-analysis.(PDF)Click here for additional data file.Figure S3INTS8-TP53INP1 region; (C–D) KCNQ1 region; (E–F) HMGA2 region.  The most strongly associated SNP in MEDIA is denoted by a purple circle and a red arrow with LD colored based on the HapMap 2 YRI data.  The index SNP is denoted by a purple circle and a blue arrow with LD colored based on the HapMap 2 CEU data.Regional plots of stage 1 meta-analysis association results in African Americans for the most strongly associated SNPs from this study and the index SNPs from previous studies. (A–B) (PDF)Click here for additional data file.Figure S4HLA-B and HLA-DQ/DR regions for  stage 1 meta-analysis association results in African Americans and HapMap 2 YRI LD data, and  stage 3 DIAGRAMv2 results in Europeans using HapMap 2 CEU LD data.  The most strongly associated SNP rs2244020 at HLA-B region from this study is denoted by a purple circle and a red arrow.  The index SNP rs9272346 from Burton PR et al (2007) Regional plots of (PDF)Click here for additional data file.Table S1Design of studies in stage 1 GWAS and stage 2a replication in African Americans.(PDF)Click here for additional data file.Table S2Clinical characteristics of study samples in stage 1 GWAS and stage 2a replication studies in African Americans.(PDF)Click here for additional data file.Table S3Genotyping methods, quality controls, imputation and statistical analysis in stage 1 GWAS and stage 2a replication studies in African Americans.(PDF)Click here for additional data file.Table S4P value≤1×10−5 from stage 1 GWAS meta-analysis (BMI unadjusted) selected for stage 2 in silico and de novo replication in African Americans and in silico replication in individuals of European ancestry from DIAGRAMv2.SNPs with (PDF)Click here for additional data file.Table S5Stage 1 GWAS meta-analysis results for index SNPs at established T2D or glucose homeostasis loci in African Americans.(PDF)Click here for additional data file.Table S6Locus-wide association at established T2D or glucose homeostasis loci in stage 1 GWAS meta-analysis in African Americans.(PDF)Click here for additional data file.Table S7BMI-adjusted association for SNPs from stage 1 GWAS meta-analysis selected for replication.(PDF)Click here for additional data file.Table S8cis-association (P<0.05) between the genome-wide significant SNPs and their proxies with the genes within 1 Mb of the associated SNPs.Expression Quantitative Trait Loci (eQTL) analysis for the genome-wide significant SNPs for T2D. Results are shown for suggestive evidence of (PDF)Click here for additional data file.Table S9TCF7L2, INS-IGF2, KCNQ1 and HMGA2.Putative regulatory SNPs predicted from the ENCODE project for the genome-wide significant SNPs and their proxies at (PDF)Click here for additional data file.Text S1Description of GWAS and replication studies.(PDF)Click here for additional data file.