S1PR2 gene have previously been linked to autosomal recessive hearing loss (DFNB68) in two Pakistani families. We describe a segregating novel homozygous c.323G>A, p.Arg108Gln pathogenic variant in S1PR2 that was identified in four affected individuals from a consanguineous five generation Iranian family.Genetic heterogeneity and consanguineous marriages make recessive inherited hearing loss in Iran the second most common genetic disorder. Only two reported pathogenic variants  in the Whole exome sequencing and bioinformatics analysis of 116 hearing loss-associated genes was performed in an affected individual from a five generation Iranian family. Segregation analysis and 3D protein modeling of the p.Arg108 exchange was performed.S1PR2 pathogenic variants presented profound hearing loss that is also observed in the affected Iranian individuals described in the current study. Interestingly, we confirmed mixed hearing loss in one affected individual. 3D protein modeling suggests that the p.Arg108 position plays a key role in ligand receptor interaction, which is disturbed by the p.Arg108Gln change.The two Pakistani families previously identified with S1PR2 and the first report outside the Pakistani population. Furthermore, we describe a novel variant that causes an amino acid exchange (p.Arg108Gln) in the same amino acid residue as one of the previously reported Pakistani families (p.Arg108Pro). This finding emphasizes the importance of the p.Arg108 amino acid in normal hearing and confirms and consolidates the role of S1PR2 in autosomal recessive hearing loss.In summary, we report the third overall mutation in The online version of this article (10.1186/s12881-018-0598-5) contains supplementary material, which is available to authorized users. Hereditary hearing loss (HL) is present in one to two per 1000 newborns . A genetS1PR2 (OMIM: 605111) encodes a 353 amino acid sphingosine-1-phosphate receptor 2 (S1PR2) and maps to the DFNB68 (OMIM: 610419) locus on chromosome 19p13.2 [S1PR2 have been implicated in congenital profound sensorineural HL without vestibular defects in two Pakistani families with recessive HL [S1PR2. 19p13.2 . VariantS1PR2 is part of the sphingosine-1-phosphate signaling (S1P) pathway and is required for normal auditory function [S1pr2 knockout mouse models have been described [S1pr2 in hearing function without other organ system involvement. Additionally, zebrafish morphants demonstrated abnormal otic vesicle and lateral line morphology, supporting a key role of this gene in auditory maintenance [function . Detailefunction . S1pr2 dfunction . To dateescribed –12. All escribed –12. Missescribed . No othentenance .S1PR2 in a proband from a large consanguineous Iranian family (E30) segregating bilateral, severe-to-profound recessive HL. We describe a homozygous pathogenic variant in S1PR2 that was identified in the first family outside Pakistan. Interestingly, this novel pathogenic variant affects the same amino acid residue as one of the previously reported mutations [S1PR2. Additionally, we model the identified amino acid residue exchange at the p.Arg108 position of S1PR2 and highlight the consequences of spatial disruption of this key amino acid with neighboring amino acid residues.In our study, whole exome sequencing of 116 hearing-associated genes disclosed the third novel pathogenic variant in utations . ClinicaThe four affected individuals  of family E30 Fig.  underwenGJB2 and STRC, respectively. Proband gDNA was subjected to whole exome sequencing. The exome library of the proband was prepared using the Nextera Rapid Capture Enrichment kit according to manufacturer’s instructions . Subsequently, the samples were 2x76 bp paired end sequenced on a NextSeq500 benchtop sequencer  with a v2 reagent kit and mapped to the human genome reference GRCh37 (hg19).Genomic DNA (gDNA) was extracted from peripheral blood leukocytes from the proband  and an in-house bioinformatics pipeline. Both bioinformatics platforms applied variant filtering against minor allele frequency >0.01 and alternate allele present at >20%. Analysis focused on missense, frameshift, nonsense and splice site variants. Pipeline data were analyzed based on the Genome Analysis Toolkit (GATK) . This inS1PR2 (NM_004230.3) variant in the single exon gene was performed using PCR-amplified gDNA from the proband (V-4) and her family members . The S1PR1 model with the ligand ML056, an analog to the natural S1P (PDB: 3v2y) [Coot [TM.The amino acid sequences of the wild type (WT) S1PR2 protein (NP_004221.3) shared 55% sequence identity with the S1PR1 protein NP_001307659.1). A homology model of the S1PR2 p.Arg108Gln exchange based on the available crystal structure of S1PR1 (PDB ID: 3v2y)  was crea7659.1. AB: 3v2y)  was usedy) [Coot  and analA five generation family E30 of Iranian descent presented four individuals  with autosomal recessive HL. Fig. The affected individuals presented HL with a prelingual onset ranging from birth to the first year of life. The HL is bilateral and severe-to-profound Fig. . IndividABRs were abnormal for individuals V-4 and V-5. ASSR thresholds for the index patient (V-4) were completely absent across all frequencies in the left ear and were detected at 90 dB each at 0.5 and 1 kHz and 110 dB each at 1 and 2 kHz. TEOAEs in the proband (V-4) were absent with 3% and 6% reproducibility for right and left ears, respectively. However, the ASSR thresholds for her younger sister (V-5) showed progressive decline Fig.  between S1PR2 (DFNB68) was identified using both bioinformatics platforms. All other variants were prioritized as benign or inadequate to cause HL in this family. Copy number variation analysis in the HL-associated genes was negative. Upon closer inspection of the exome dataset using GensearchNGS, the S1PR2 c.323G>A pathogenic variant was found to reside in a 1.65 Mb run of homozygosity on chromosome 19p13.2 . An in-house exome database filtered for S1PR2 variants in the nearly 300 families with HL who are included in our on-going exome projects did not disclose additional putative pathogenic variants in S1PR2.The proband V-4 was subjected to whole exome sequencing and bioinformatics analysis that included 116 HL-associated genes. Bioinformatics analysis revealed over 1600 unfiltered variants that were filtered after pipeline analysis to three heterozygous variants and one homozygous variant in autosomal recessive HL genes, as well as one heterozygous variant in an X-linked recessive HL gene and two heterozygous variants in autosomal dominant HL genes. A homozygous c.323G>A, p.Arg108Gln variant in the gene S1PR2 had a mean depth of 44-fold and was covered with over 80 reads at the c.323 position. Overall, 95% of all HL gene capture regions were covered with a mean depth of at least 10×. 83.4% of S1PR2 achieved a 10-fold coverage. The c.323G>A, p.Arg108Gln variant results in a likely pathogenic amino acid exchange according to pathogenicity prediction tools . The variant was not archived in ClinVar, DVD, HGMD, ExAC and gnomAD databases  and amino acid up to tetraodon (considering 11 species). The physiochemical difference between the arginine and glutamine amino acid residue exchanges is small  . Nevertheless, the c.323G>A provokes a small change in the 3’ splice performance in two of the five splice prediction tools in Alamut (data not shown). A novel splice site that is the result of the c.323G>A variant creates a novel 3’ splice acceptor site with a splicing prediction score of 87.1 (reference range: 0-100) in Human Splicing Finder and 2 (reference range: 0-16) in MaxEntScan. The functional impact of this predicted cryptic splice site activation was not tested. Furthermore, the variant affects one of the 67 CpG sites in the single coding exon of ing Fig.  and segring Fig. . All affS1PR2.Interestingly, the same c.323 position has been reported with a different homozygous nucleotide exchange (c.323G>C) in a family with congenital profound sensorineural HL and lower limb deformities . This vaBased on the S1PR1 crystal structure, we designed a homology model of S1PR2 to compare the structural outcome of p.Arg108Gln with the previously published p.Arg108Pro amino acid exchange. The WT protein structure has seven transmembrane helices (TM1-7). Together with extracellular loops 1 and 2 (ECL1 and ECL2), the N-terminal helix I Fig. , green cIn a resting state, without interaction of S1P, Arg108 coordinates a hydrogen bond (H-bond) network including residues Leu92, Thr97, Gln104, Trp105 and Glu109 Fig. . The subS1PR2 variant is involved in this first reported observation of S1PR2 HL in a large Iranian family. Nevertheless, variants in S1PR2 seem to be quite rare considering that no further putative pathogenic alleles were present in our cohort of approximately 300 families including over 90 Iranian families.The prevalence of inherited HL in Iran is higher than in Western countries , which i+ for electrochemical gradient maintenance [S1PR2 are thought to affect S1P ligand activation or non-formation of the S1PR1-G protein complex [S1P, a lysophospholipid intermediate, binds to the N-terminal ECL surface of S1PR2  and phosntenance , 41. Pat complex .The identified c.323G>A p.Arg108Gln) pathogenic variant in family E30 most likely affects the function of S1PR2. In a resting state, the novel amino acid residue exchange could potentially influence the receptor by loss or gain of specific H-bonds Fig. . WhereasGln pathoS1pr2 can be seen at postnatal day 14 of knockout mice. By postnatal day 22, hair cell and spiral ganglion neuron degeneration can be observed and corresponds with profound deafness [s1pr2 homolog revealed abnormalities in the otic vesicle, as well as the lateral line hair cells and neuromasts. Morphant semicircular canals showed structural defects. The hair cell and otic vesicle pathologies mirrored the cochlear and vestibular deficits described in the various mouse models [Pathological changes in the stria vascularis by mutated deafness . Progresdeafness , whereasdeafness . A knocke models , 13.S1pr2 is expressed in the inner and outer hair cells of mice but hair cell morphology is normal at hearing onset in the many mouse mutants published to date [S1pr2 mutant mice may be the result of two pathological processes that are due to disruption of EP and an accumulation of reactive oxygen species that lead to a degeneration of the cochlea. The progressive hair cell dysfunction is likely a secondary effect; however, whether mutated S1PR2 directly causes pathological lesions in hair cells cannot be excluded [Interestingly,  to date –12. Hairexcluded , 42. Wheexcluded , 38.The severe-to-profound HL in the affected individuals of family E30 was apparent at an early age. This severe HL is similar to families DEM4154 and PKDF1400


Human adenoviruses are small double stranded DNA viruses that provoke vast array of human diseases. Next generation sequencing techniques increase genomic data of HAdV rapidly, which increase their serotypes. The complete genome sequence of human adenovirus shows that it contains large amount of proteins with unknown cellular or biochemical function, known as hypothetical proteins. Hence, it is indispensable to functionally and structurally annotate these proteins to get better understanding of the novel drug targets. The purpose was the characterization of 38 randomly retrieved hypothetical proteins through determination of their physiochemical properties, subcellular localization, function, structure and ligand binding sites using various sequence and structure based bioinformatics tools.Function of six hypothetical proteins P03269, P03261, P03263, Q83127, Q1L4D7 and I6LEV1 were predicted confidently and then used further for structure analysis. We found that these proteins may act as DNA terminal protein, DNA polymerase, DNA binding protein, adenovirus E3 region protein CR1 and adenoviral protein L1. Functional and structural annotation leading to detection of binding sites by means of docking analysis can indicate potential target for therapeutics to defeat adenoviral infection.The online version of this article (10.1186/s13104-017-2992-z) contains supplementary material, which is available to authorized users. Human adenoviruses are non-enveloped dsDNA viruses of almost 35 kb in size . HAdV caMastadenovirus in which all the human adenoviruses are categorized and further divided into different strains [Seven known Human adenoviruses species from HAdV-A to HAdV-G are constitute of the genus  strains . Now 67  strains , 9.After an immense effort 50–60% genes have a known function in most of completely sequenced genomes. Number of genes having unknown functions called as hypothetical protein are present in each organism’s genome . To undeIn-silico strategies to annotate the hypothetical proteins are cost effective and fast enough to explore their function. In this study, multiple algorithm based software’s have been used for the prediction of hypothetical protein function that may lead to the identification of novel pharmacological targets for screening, drug discovery and designing for the treatment of HAdV infections .Proteins having unknown function of Human adenovirus were taken from UniProt , 13. RanAnalysis of physiochemical properties of all HPs was done by online server ExPASy’s Protparam tool . This seTo predict the cellular function of a protein it is important to get information about its sub-cellular localization i.e. a protein can be present in outer membrane, inner membrane, periplasm, extracellular space or in cytoplasm . Sub-celMost basic step in the function prediction of a protein is looking for its structural homologs in different available genomics and proteomics based databases. Popular bioinformatics tool BLASTp was used for this purposes , 24.For precise function annotation, various tools like SVMport, ProtNet , 26, PfaFor prediction and validation of three dimensional I-TASSAR (Iterative Threading Assembly Refinement) , 41 RamaFunctions of proteins based on structural analysis are considered more acceptable as compared to sequence based function annotation, because homologous proteins show more conserved structures in evolution than sequences . For thiRandom selection of 38 hypothetical proteins belonging to eight different types of HAdV was carried out from UniProt .Additional file 4: Table S4. This table details list of annotated function of 38 human adenovirus using BLASTp, SMART, INTERPROSCAN and MOTIF.Additional file 5: Table S5. This table presents list of functionally annotated domain and motifs of HPs 38 s from human adenovirus by CATH, SUPERFAMILY, Pfam, CDART, ProtNet and SVMprot.Additional file 6: Table S6. This table presents ligand binding sites prediction of hypothetical proteins of human adenovirus.Additional file 7: Table S7. Sequence and structure motif prediction by pro-func.Additional file 8: Figure S1. 3D structure of hypothetical protein P03269 predicted from I-TASSER.Additional file 9: Figure S2. Evaluation of 3D structure of hypothetical protein P03269 through Ramachandran Plot.Additional file 10: Figure S3. 3D structure of hypothetical protein P03261 predicted from I-TASSER.Additional file 11: Figure S4. Evaluation of 3D structure of Hypothetical Protein P03261 through Ramachandran PlotAdditional file 12: Figure S5. 3D structure of hypothetical protein P03263 predicted from I-TASSER.Additional file 13: Figure S6. Evaluation of 3D structure of hypothetical protein P03263 through Ramachandran Plot.Additional file 14: Figure S7. 3D structure of hypothetical protein Q83127 predicted from I-TASSER.Additional file 15: Figure S8. Evaluation of 3D structure of hypothetical protein Q83127 through Ramachandran Plot.Additional file 16: Figure S9. 3D structure of hypothetical protein Q1L4D7 predicted from I-TASSER.Additional file 17: Figure S10. Evaluation of 3D structure of hypothetical protein Q1L4D7 through Ramachandran Plot.Additional file 18: Figure S11. 3D structure of hypothetical protein 16LEV1 predicted from I-TASSER.Additional file 19: Figure S12. Evaluation of 3D structure of hypothetical protein I6LEV1 through Ramachandran Plot.


Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity.Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite  Plasmodium falciparum is an apicomplexan parasite that causes the most virulent form of human malaria. All of the pathology of the disease is caused by the asexual blood-stage parasite, which develops and multiplies within red blood cells (RBC). Intracellular development through the trophozoite and schizont phases results in a multinucleate cell and then cell division produces individual haploid merozoites. This process is accompanied by the elaboration of apical organelles such as micronemes and rhoptries, and an inner membrane complex (IMC) that forms part of the parasite’s surface pellicle. Following release (or egress) from the schizont, the extracellular merozoites invade new RBC and the cycle continues. The invasion of RBC is a crucial step in the parasite life-cycle and, because the parasite's exposed surface antigens are especially susceptible to immune attack, rapid host cell entry is important. Interactions between parasite and host cell proteins are very important in this process.The full life-cycle of this parasite is complex and includes other extracellular stages that are motile and that invade host cells and tissue. The motile sporozoite and ookinete stages move by a process called gliding motility, which is powered by a multi-molecular complex called the glideosome. In the current model, the glideosome, containing a class XIV myosin, myosin A (MyoA); a myosin light chain called the MyoA tail domain-interacting protein (MTIP); and glideosome-associated proteins (GAPs) such as GAP50, GAP45, and GAP40 , is locain vitro with aldolase  ATP was added to 10μl 1mM ATP, and 2μl of this mixture added to each sample to initiate the kinase reaction. Following incubation at 30°C for 3 hours, samples were mixed with SDS-PAGE loading buffer (1:1 vol/vol), and heated to 90°C for 5 minutes. Each sample was loaded in two halves on a 10% Bis-Tris NuPAGE gel using MOPS running buffer. The gel was fixed, dried and exposed to Kodak Biomax MR film to visualize the radiolabelled proteins, or the proteins were visualised by Coomassie Brilliant Blue staining to ensure that equivalent amounts of protein were loaded.Phosphorylation assays were performed using 10 μM recombinant CDPK1 , PKA (PrZipTip® Pipette Tips (Millipore) containing either C18 or C4 according to the size of the protein to be desalted. For CID analysis, the eluted proteins in 70% (vol/vol) acetonitrile (ACN) and 0.1% (vol/vol) acetic acid were injected into the mass spectrometer using a 10 μl min-1 flow rate. Phosphorylation sites were determined using stand-alone nanospray on an LTQ Orbitrap Velos (Thermo Fisher Scientific). Nanospray needles  were operated at 1.6 kV and collision induced dissociation MS/MS was performed in the ion trap and recorded in the Orbitrap. Spectra were acquired over a collision energy range of 20-30eV at a resolution of 100k. The averaged MS/MS spectrum was then deconvoluted using Xtract and interpreted using Prosight (https://prosightptm.northwestern.edu/).Non-phosphorylated, phosphorylated and biotinylated polypeptides were analysed by electrospray (ES)-mass spectrometry and collision-induced dissociation (CID) fragmentation pathway analysis. The samples (100 pmol) were first desalted using P10 -1 of endopeptidase, overnight at 32°C. Digestion in solution was performed in a 20 μl final reaction volume with a molar ratio of 1:100 enzyme:protein in 5 mM ammonium bicarbonate, with overnight digestion at 32°C. One to ten microlitres of peptide solution acidified to a final concentration of 0.1% TFA was loaded at 5 μl min-1 onto a 2 mm x 100 μm Acclaim Pepmap C18 trap column attached to an Ultimate 3000 nanoRSLC HPLC (Thermo Scientific), then peptides were eluted at 0.22 μl min-1 through a 50 cm x 75 μm Acclaim Pepmap C18 column. Starting with Buffer A , a gradient from 9% to 25% buffer B  over 37 minutes, and 25% to 40% buffer B over 18 minutes was used; followed by a short gradient to 100% B. Eluant was introduced into a LTQ Orbitrap Velos Pro (Thermo Scientific) using a Proxeon NanoES source (Thermo Scientific) and a 30 μm ID stainless steel emitter operated at 2 kV. The Orbitrap was operated in “Data Dependent Acquisition” mode with a survey scan at a resolution of 60,000 from m/z 300–1500, followed by MS/MS in the LTQ of the top 6–12 ions. Dynamic exclusion was used with a time window of 40 s. The Orbitrap charge capacity was set to a maximum of 106 ions in 10 ms, whilst the LTQ was set to 104 ions in 100 ms. Raw files were processed using Proteome Discoverer (PD) 1.3 (Thermo Scientific) with Mascot 2.4  as the search engine against the appropriate protein fasta database. A decoy database of reversed sequences was used to filter the results at a false detection rate of 1%. Label free quantitation was achieved using the precursor-ion quantitation module of PD.For protein analysis, peptides were generated by in-gel or in-solution digestion using sequencing-grade modified endopeptidases, trypsin, Asp-N or chymotrypsin (Promega). The in-gel digestion was performed after first resolving proteins by SDS-PAGE and subsequent staining using the Colloidal Blue staining kit (Invitrogen), according to the manufacturer’s instructions. The proteins bands were excised and both SDS and stain removed with 100% (vol/vol) ACN, and 5 mM ammonium bicarbonate containing 50% (vol/vol) ACN, for 30 min at room temperature. The proteins were then reduced using 20 mM DTT for 1 hour and alkylated with 5 mM iodoacetamide for 20 min at room temperature. The gel slices were first dehydrated in 100% (vol/vol) ACN and then the proteins digested in 20 μl final volume in 5 mM ammonium bicarbonate using 2 ng μlmrw) Secondary structure content was estimated using methods described elsewhere [Far-UV CD spectra were recorded on a Jasco J-715 spectropolarimeter fitted with a cell holder thermostatted by a PTC-348 Peltier unit. All CD measurements were made in 20 mM Tris (pH 8) at 20°C using fused silica cuvettes with 1 mm path length . The spectra were typically recorded with 0.1 nm resolution and baseline corrected by subtraction of the appropriate buffer spectrum. CD intensities are presented as the CD absorption coefficient calculated on a mean residue weight basis  at 25°C in 96-well microplates, as described previously . All assFor competition experiments, for example of MTRAP in the interaction between AMA1 or RH4 and aldolase, the Octet Red system was also used. Biotinylated AMA1 or RH4 peptide was loaded onto the streptavidin coated sensor and following washing incubated with 20 or 70 μM aldolase with (or without) increasing concentrations of unlabelled competitor MTRAP CTD peptide. Following incubation the data were collected and analysed as described above. The experiments were performed in duplicate.P. falciparum MTRAP with a GFP tag situated distal to the transmembrane (TM) sequence and proximal to the CTD was placed into the pHH4 vector [mtrap locus in the parasite by homologous recombination (see CCCGGGTCGAAAAATGCTAGAG GTTATAGAGG (forward) and GGCCTAGGTTTTTCTTTACGTAAGAAATAG (reverse) to incorporate XmaI and AvrII restriction enzyme sites (underlined). The sequence coding for MTRAP CTD was synthesized in a recodonised form: TATTTTCTGCGCAAAGAAAAAACCGAAAAAGTGGTGCAGGAAGAAACCAAAGAAGAAAATTTTGAAGTTATGTTTAATGATGATGCCCTGAAAGGCAAAGATAATAAAGCCATGGATGAAGAAGAATTTTGGGCACTGGAATAA (GeneArt®). The GFP sequence was obtained from the pHH4 plasmid and fused to MTRAP CTD sequence by PCR amplification using the primers: CCCCTAGGAGTAAAGGAGAAGAACTTTTCAC, TTTTTTCTTTGCGTTTG TATAGTTCATCCATGCCATGTGTAATCCC, and GGCCGCGGTTATTCCAGTGCCC to introduce AvrII and SacII restriction enzyme sites. These DNA fragments were then incorporated upstream of the P. berghei dhfr 3’ UTR in the pHH4 plasmid and Sanger sequencing (Beckman Genomics) was used to confirm the correct sequence assembly.Sequence coding for 4 vector  for insetion see . The regP. falciparum 3D7 ring stage parasites were transfected with 100 μg of plasmid DNA and cultured continuously under a WR99210 (2.5 nM) drug selection cycle for 4 cycles over 12 weeks. After each cycle parasite cultures were examined for genomic integration by PCR and for GFP expression by fluorescence microscopy. PCR was performed with the following primers: F1 (GTGGGAGAGACATTTAAGAAGTAATA) and R1 (CTCGGTGGGGCTTTAAATAATG) targeting sequences upstream and downstream of endogenous mtrap, F1 and R2 , and F1 and R3 . Following detection of integration the parasites were cloned by limiting dilution. A schematic representation of the parasite integration, diagnostic PCR and western blot is presented in To incorporate the modified gene, P. falciparum asexual blood stages were cultured in human RBCs, and analysed by fluorescence microscopy and western blotting, using preparations of schizonts and merozoites purified as described previously [eviously . GFP-tageviously .S1 Fighttps://prosightptm.northwestern.edu/) or Proteome Discoverer (PD) 1.3 (Thermo Scientific).Phosphorylation sites were interpreted from the mass data using either Prosight ( A. MTRAP phosphorylation (MTRAP_1 to MTRAP_6) interpreted using Prosight. There are only two potential phosphorylation sites and each of these threonines can be phosphorylated. B. AMA1 phosphorylation (AMA1_1 to AMA_5) interpreted using PD1.3. Phosphorylation was by CDPK1 (AMA1_1 to AMA1_3) and PKA (AMA1_4 and AMA1_5).The data are consistent with phosphorylation of Ser610 and not of the other 6 serine/threonine residues in the sequence. C. RH1 phosphorylation by CDPK1 interpreted using Prosight. The method did not resolve whether Ser2950 or Thr2951 was phosphorylated. D. RH4 phosphorylation by CK2 (RH4_1 to RH4_7) interpreted using PD1.3. The data are consistent with phosphorylation of either Ser1667 or Ser1674 with a very small amount of phosphorylation of both residues. E. EBA140 phosphorylation by CDPK1 (EBA140_1 to EBA140_2) interpreted using Prosight. No phosphorylated residues were identified unambiguously. F. EBA175 phosphorylation by CDPK1 (EBA177_1 to EBA175_2) interpreted using Prosight. Phosphorylation of both Thr1483 and Ser1489 was detected. G. EBA181phosphorylation by CDPK1 (EBA181_1 to EBA181_21) interpreted using PD1.3. Phosphorylation of both Ser1523 and Ser1528 was detected.(PDF)Click here for additional data file.S2 FigA. Phosphorylation of recombinant MTRAP peptide by protein kinase A. The MTRAP peptide  was biotinylated  and then phosphorylated using PKA (lower panel). The peaks corresponding to the protein and its phosphorylated forms are indicated. B. Phosphorylation of synthetic biotinylated AMA1 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). The non-phosphorylated peptide  and a single phosphorylated form (6968.4) were detected. C. Phosphorylation of synthetic biotinylated RH1 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). Only the non-phosphorylated peptide  was detected after incubation with PKA and an additional single (6619.9) phosphorylated form was detected following incubation with CDPK1. D. Phosphorylation of synthetic biotinylated RH4 peptide by CDPK1 (upper panel), protein kinase A (middle panel) and casein kinase 2 (lower panel). Only the non-phosphorylated peptide  was detected after incubation with CDPK1 or PKA and a single (8386.2) phosphorylated form was detected following incubation with CK2. E. Phosphorylation of synthetic biotinylated EBA140 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). Only the non-phosphorylated peptide  was detected after incubation with PKA. F. Phosphorylation of synthetic biotinylated EBA175 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). Only the non-phosphorylated peptide  was detected after incubation with PKA and a single (7243.2) phosphorylated form was detected following incubation with CDPK1. G. Phosphorylation of synthetic biotinylated EBA181 peptide by CDPK1 (upper panel) and protein kinase A (lower panel). Only the non-phosphorylated peptide  was detected after incubation with PKA and single (7094.9) and dual (7173.9) phosphorylated forms were detected following incubation with CDPK1.(PDF)Click here for additional data file.


The PA N-terminal domain (PA-Nter) is essential for viral transcription and replication. Here we identified PA-Nter substitutions A37S, I61T, V63I and V100A in recently emerged avian influenza A viruses (IAVs) with potential effect on virus pathogenicity and/or host adaptation. We introduced the identified PA-Nter substitutions into avian H7N7 IAV by reverse genetics. Our results showed that single substitution V63I and combined substitutions, I61T/V63I and A37S/I61T/V63I/V100A (Mfour), significantly increased virus growth capacity in mammalian cells. Meanwhile, these substitutions conferred higher virus transcription/replication capacity by producing more mRNA, cRNA and vRNA. Consistently, the polymerase activity and the endonuclease activity were enhanced by these PA-Nter substitutions. Notably, substitutions V63I and Mfour strongly increased virus replication and virulence in mice. Collectively, our findings demonstrated that the PA-Nter substitutions V63I and Mfour enhanced IAV pathogenicity through modification of the polymerase activity and the endonuclease activity, which added to the evolving knowledge of IAV virulence determinants. In the past decades, more than ten different subtypes of avian influenza A viruses (IAVs) have overcome species barriers and transmitted to humans, posing great threats to the public health8et al. found PA-Nter substitutions D108A and K134A completely blocked the endonuclease activityet al. also confirmed a number of PA-Nter mutations  that inhibited the endonuclease activity17181920in vitroin vivo when combined with mutation PB2-E627K20The pathogenicity of avian IAVs are polygenic, being determined in part by the constellation of virus genes1214in vitro. We further investigated the effect of the substitutions V63I and Mfour on virus replication and virulence in mice. Notably, we demonstrated that the PA-Nter substitutions, V63I or Mfour, conferred enhanced virus pathogenicity in vitro and in vivo. Our study may help to understand the pathogenicity of the emerging avian IAVs and other IAVs carrying these substitutions, which adds to the evolving knowledge of IAV virulence determinants and facilitates the assessment of pandemic risks.In this study, we identified PA-Nter substitutions A37S, I61T, V63I and V100A in recently emerged avian IAVs with potential effect on virus pathogenicity and/or host adaptation by comprehensive bioinformatics analyses. To investigate the biological significance of these PA-Nter substitutions, we selectively generated mutant viruses of A/Netherlands/219/2003 (H7N7) harboring single or combined substitutions, I61T, V63I, I61T/V63I, A37S/V100A and A37S/I61T/V63I/V100A (Mfour) by reverse genetics. Virus growth capacity, transcription/replication capacity, the polymerase activity and the endonuclease activity of these mutants were compared with those of the wild type By sequence alignment and analyses, we identified amino acid variations at positions 37, 61, 63 and 100 of PA-Nter among influenza A viruses (IAVs) from human infections . We foun678P = 0.0159), 3.2-fold (P = 0.0025) and 2.2-fold (P = 0.0286) higher than those of WT-H7N7 at 9, 12 and 24 hours post-infection (hpi), respectively. Therefore, the substitution Mfour (A37S/I61T/V63I/V100A) enhanced virus growth of avian H7N7 IAV in mammalian cells.As PA-Nter residues of H7N9 IAV differed from those of WT-H7N7 exclusively at positions 37, 61, 63 and 100, we first asked whether these four substitutions affected virus growth capacity. The plasmid-based reverse genetics of avian H7N7 IAV was established . RecombiNext, we generated additional mutant viruses to delineate the effect of PA-Nter substitutions. Recombinant viruses carrying the single substitutions I61T and V63I were obtained by reverse genetics . As the P < 0.05), whereas the titer of the A37S/V100A mutant virus was lower than that of WT-H7N7 (P < 0.001). At the same time, the titer of the I61T mutant virus was not statistically different from that of WT-H7N7. Thus, our data indicated that V63I might be able to override the effect of suppressive residue(s) and play a predominant role in determining the virus growth capacity. Collectively, the PA-Nter substitutions V63I, I61T/V63I and Mfour (A37S/I61T/V63I/V100A) increased virus growth capacity in mammalian cells, whereas the substitution A37S/V100A posed a decreased effect.To evaluate the impact of the substitutions on virus growth, we inoculated recombinant viruses into MDCK cells at an MOI of 1 and harvested supernatants at 8 hpi. As shown in P < 0.05). Among these substitutions, V63I resulted in the highest amount of mRNA, cRNA and vRNA at 6 hpi (P < 0.05) (P = 0.0120), cRNA (P = 0.0378) and vRNA (P < 0.001) when compared with those of WT-H7N7 at 4 hpi  . The I61at 4 hpi . Taken tP < 0.001) (P < 0.001)  . In cont< 0.001) . Hence, E. coli. Purified PA-Nter proteins were obtained in the second eluates (E2) , whereas the PA-Nter I61T protein showed lower endonuclease activity than that of WT-H7N7 (P = 0.0119) when compared to that of WT-H7N7   and 4B a WT-H7N7 . Thus, tin vitro. At the same time, V63I was indicated to pose a dominant effect on enhancing the virulence among the tested substitutions. Thus, we next selectively investigated the effect of these two substitutions on virus pathogenicity in vivo. BALB/c mice were intranasally inoculated with WT-H7N7, V63I mutant virus, Mfour mutant virus or PBS. As shown in P < 0.01). All the V63I and Mfour mice succumbed to the infection by day 9 while 20% of the WT-H7N7 mice survived the infection (P < 0.05), while the Mfour exhibited significantly higher lung viral titers on day 5 post-infection (P = 0.0065)  was prevalent in the majority of recently emerged IAVs, which increased the virus growth and transcription/replication capacity nfection . Moreove 0.0065) . Thus, wThe 3D models of WT-H7N7 and Mfour (A37S/I61T/V63I/V100A) PA-Nters were generated and validated. Molecular docking was performed to identify the potential differences between the PA-Nter structures of WT-H7N7 and Mfour. As shown in In this study, we identified PA-Nter substitutions at positions 37, 61, 63 and 100 in the recently emerged avian influenza A viruses (IAVs) that played key roles in promoting virus replication and might facilitate virus transmission to human hosts. We found that the PA-Nter substitution V63I alone and combined substitutions, I61T/V63I and Mfour (A37S/I61T/V63I/V100A), enhanced virus growth capacity when compared to that of wild type avian H7N7 IAV in mammalian cells. At the same time, these substitutions potentiated viral transcription and replication by producing more virus mRNA, cRNA and vRNA in infected cells. Furthermore, the polymerase activity and the endonuclease activity were augmented by these PA-Nter substitutions. Notably, the V63I and Mfour substitutions enhanced virus growth and virulence in mice. Collectively, our data demonstrated that the substitution V63I alone and the combined substitution Mfour (A37S/I61T/V63I/V100A) increased viral pathogenicity of avian H7N7 IAV through modification of the polymerase activity and the endonuclease activity.Genetic determinants in virus genes i.e. HA and NS3218343536We identified PA-Nter substitutions at positions 37, 61, 63 and 100 in recently emerged IAVs, including the majority of H7N9 and H5N6 IAVs isolated in 2015. Avian H7N9 IAV has caused a large number of severe human infections in China since 201322et al. introduced mutations into the PA-Nter domain that inactivated the endonuclease activity and showed that these mutations might be involved in the positioning of RNA substrates in the endonuclease active sitein vitro and in vivo. This observation can be reconciled with the potential structural changes of the expressed PA-Nter domain, the involvement of the rest of the polymerase complex in viral transcription and replication, or the direct or indirect interactions of these substitutions with specific host cellular factorsVirus mRNA transcription is primer-dependent, which requires caps snatched from the host cells in the initiation step1114et al. demonstrated that residues at the PA-Nter flexible loop region suppressed cellular protein synthesisMoreover, the flexible loop region of PA-Nter (residues 53–73) is typically in a disordered structure and its function has been poorly understoodet al. reported that PA-Nter substitutions S37A and A100V slightly enhanced the virulence of avian H7N9 IAV in mice, though the differences were not statistically significantin vitro through decreasing the polymerase activity in avian H7N7 IAV. However, combined with the other two PA-Nter substitutions, the A37S/I61T/V63I/V100A increased the virus pathogenicity in vitro and in vivo. These findings indicate that the PA-Nter substitution A37S/I61T/V63I/V100A presented by the recently emerged IAVs contributes to an enhanced pathogenicity, though certain residues may pose an attenuated effect.Yamayoshi in vitro and in vivo. The identification of these substitutions as pathogenic and human adaptation determinants may add to the current mutational analysis of the role of PA and aid in the assessment of pandemic potential of avian H7N7 IAV as well as other IAV subtypes carrying these substitutions in humans.In conclusion, we demonstrated that the PA-Nter substitutions V63I and Mfour (A37S/I61T/V63I/V100A) increased virus growth capacity and pathogenicity 2. Human embryonic kidney (HEK) 293-T cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS and 1% P/S. Avian influenza A virus (IAV) strain A/Netherlands/219/2003 (H7N7) (WT-H7N7) and mutant viruses were cultured in MDCK cells supplemented with 1% P/SMadin-Darby canine kidney (MDCK) cells were grown in Eagle’s minimum essential medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (P/S) at 37 °C with 5% COhttp://platform.gisaid.org) (isolate IDs available on request). Multiple sequence alignments were performed using the MEGA 6.06 software. The residues in the PA N-terminal domain  were inspected and analyzed among different subtypes to identify the varied ones that might associate with virus pathogenicity.Full-length PA protein sequences of various IAV subtypes isolated from human infections were retrieved from the website Global Initiative on Sharing All Influenza Data (GISAID) (The eight gene segments of avian IAV strain A/Netherlands/219/2003 (H7N7) were cloned into expression vector pHW2000, respectivelyPA-Nter substitutions were introduced into plasmid pHW2000-PA (WT-H7N7) using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies).The eight plasmids possessing virus gene segments were mixed with ScreenFect A transfection reagent (InCella) according to the manufacture’s protocol and then added into the co-cultured 293-T and MDCK cells in the 12-well plates. The medium was replaced with Opti-MEM (Life technologies) supplemented with 1% P/S at 6 hours post transfection. The transfected cells were cultured at 37 °C for four days to rescue WT-H7N7 and mutant viruses. The viruses rescued were verified by sequencing.Growth capacity of WT-H7N7 and mutant viruses were investigated. Briefly, the viruses were inoculated into MDCK cells at a multiplicity of infection (MOI) of 1. Cell-free supernatants were harvested at the indicated time points and titrated in MDCK cells by plaque assayet al.et al.A two-step RT-qPCR assay was performed to quantify the abundance of viral mRNA, cRNA and vRNA, which would reflect viral transcription and replication capacity, as reported by Kawakami ® 96 (Roche) machine. The primers targeting at the specific tags added in the cDNAs were utilized (primer sequences available on request). Briefly, 5 μl of a 1:10 diluted cDNAs were mixed with 10 μl of 2 × FastStart SYBR Green I Master (Roche), 1 μl of forward primers (10 μM), 1 μl of reverse primers (10 μM) and 3 μl of sterile water. Virus RNA levels were normalized with GAPDH mRNA levelReal time qPCR was carried out on the LightCycler® 3000 Transfection Reagent (Invitrogen). The cell lysates were harvested at 24 hours post transfection. Luciferase activity was measured on a Victor X3 multilabel plate reader (Perkin Elmer) by using Bright-Glo™ Luciferase Assay System (Promega). Fluorescence signals were also detected as an internal control for the transfection efficiency. The luciferase readings were normalized with the fluorescence readings.The polymerase activity of WT-H7N7 and mutants were investigated as previously described6-tag (Novagen). The resulted plasmids were then transformed into E. coli strain BL21 (DE3) for protein expression50E. coli, expressed, purified and used as the negative control. Purified proteins were confirmed by 10% SDS-PAGE gel and Western blotThe DNA fragments of PA-Nter (residues 1–196) were amplified from pHW2000-PA plasmids (with or without the substitutions) and cloned into pET-32a (+) expression vector containing a His2, pH 7.8) at 37 °C. Fluorescence signals were released after the cleavage of the DNA substrates by the PA-Nter proteins. The fluorescence signals were measured at the indicated time points on the Victor™ X3 multilabel plate reader (Perkin Elmer).Purified PA-Nter proteins were quantified using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Fluorescence-based endonuclease assay was performed as described previously50Six to eight-week-old female BALB/c mice (11 mice/group) were anesthetized by intraperitoneal injection of ketamine-xylazine (50/5 mg/kg) and intranasally inoculated with 120 plaque-forming units (PFU) of the indicated viruses in a volume of 20 μl. Five BALB/c mice were anesthetized and intranasally inoculated with 20 μl of PBS as a negative control. Mouse body weight and survival were monitored daily for up to 14 days. The mice that lost more than 20% of their initial body weight were humanely euthanized. To evaluate virus replication in mice, 3 mice in each group were euthanized on days 3 and 5 post-infection and mouse lungs were excised. Lung tissues were homogenized in MEM medium and virus titers in the supernatants were titrated in MDCK cells by plaque assay44http://services.mbi.ucla.edu/SAVES/) with PROCHEKhttp://xylian.igh.cnrs.fr/bin/align-guess.cgi) with the template sequences. As PA-Nter proteins retained the endonuclease activity with Mn2+ ions2+ ions, PDB files of which were retrieved from 4E5G (the PDB structure file was retrieved from RCSB data bank) using PDB editor. The PA-Nter models of WT-H7N7 and Mfour were then docked with Mn2+ ions at the active sites of receptor by using patchdock algorithm2+ ions were docked with 3D model of a single-stranded DNA sequence (5′-TCTCTAGCAGTGGCGCC-3′), which was experimentally proved to be a substrate of the enzymehttp://structure.usc.edu/make-na/server.html)56The 3D models of WT-H7N7 and Mfour PA-Nter were prepared by Swiss Model algorithmt-test, or one-way ANOVA followed with a Dunnett’s test, or Log-rank (Mantel-Cox) test with the GraphPad Prism 6 software package  were used for data analysis. P < 0.05 was considered significantly different.Two-tailed Student’s How to cite this article: Hu, M. et al. Amino acid substitutions V63I or A37S/I61T/V63I/V100A in the PA N-terminal domain increase the virulence of H7N7 influenza A virus. Sci. Rep.6, 37800; doi: 10.1038/srep37800 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Furthermore, expression levels of ABA biosynthesis genes ABA3 and NCED3 were repressed by HAT1 directly binding to their promoters, resulting in the ABA level was increased in hat1hat3 and reduced in HAT1OX lines. Further evidence showed that both protein stability and binding activity of HAT1 was repressed by SnRK2.3 phosphorylation. Overexpressing SnRK2.3 in HAT1OX transgenic plant made a reduced HAT1 protein level and suppressed the HAT1OX phenotypes in ABA and drought response. Our results thus establish a new negative regulation mechanism of HAT1 which helps plants fine-tune their drought responses.Drought is a major threat to plant growth and crop productivity. The phytohormone abscisic acid (ABA) plays a critical role in plant response to drought stress. Although ABA signaling-mediated drought tolerance has been widely investigated in Arabidopsis thaliana, the feedback mechanism and components negatively regulating this pathway are less well understood. Here we identified a member of Arabidopsis HD-ZIP transcription factors HAT1 which can interacts with and be phosphorylated by SnRK2s.  hat1hat3 mutant showed ABA-hypersensitive and drought-tolerant phenotypes, whereas the HAT1-overexpressing lines were insensitive to ABA and less tolerant to drought. Furthermore, we found SnRK2.3 kinase, a positive component of ABA signaling, interacts with and phosphorylates HAT1 to destabilize and suppress its binding activity. Overexpression of SnRK2.3 reduces HAT1 protein level and inhibits HAT1OX phenotypes in ABA and drought responses. Our results revealed a HAT1-mediated negative regulatory mechanism in attenuating the ABA signaling and drought response.Drought stress is a key environmental factor that severely reduces crop yield all over the world. The phytohormone abscisic acid (ABA) is known to mediate drought responses through regulating drought-responsive genes expression and stomatal closure, but the mechanisms that negatively regulate this process and prevent the adverse effects of excess drought responses on plant growth is less well studied. Here, we show that a HD-ZIP II transcription factor HAT1 negatively regulates ABA-mediated drought responses through suppressing ABA biosynthesis and signaling. The  As sessile organisms, plants need to respond and adapt to environmental stress to survive adverse conditions. Plants respond and adapt to stresses through a complex network of factors involved in stress hormone signaling and regulation of gene expression. The phytohormone abscisic acid (ABA) plays a key role in plant responses to biotic and abiotic stress, in particular drought and salinity –3.Since the discovery of ABA receptors, PYRABACTINRESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORYCOMPONENTS OF ABA RECEPTORS (RCAR) ,5, a corHowever, in many cases, stress adaptation is exchanged for growth and productivity, therefore, it is necessary for plants to develop a resilient system to obtain the optimal trade-off for survival and growth. To this end, plants use elaborate mechanisms associated with posttranscriptional modulation  and postThe homeodomain-leucine zipper protein (HD-ZIP) family constitute a large family of transcription factors that are unique to plants and is divided into four subfamilies (HD-ZIP I–IV) on the basis of the additional conserved domains, structures and physiological functions –23. HD-ZHAT1 and HAT3 were repressed by ABA and drought, and the double mutant hat1hat3 displayed a reduced ABA sensitivity and enhanced drought tolerance phenotype that was stronger than the single mutants alone. HAT1-overexpressing transgenic plants exhibit a hyposensitive response to ABA and drought. Furthermore, we found that HAT1 physically interacts with and can be phosphorylated by SnRK2.3 in vitro and in vivo. SnRK2.3 phosphorylation of HAT1 decreased its protein stability and binding activity. Overexpressing SnRK2.3 in HAT1OX transgenic plant can suppress its phenotype in ABA and drought responses. Therefore we identified a new substrate of SnRK2.3 and established a novel negative regulation mechanism by which plants can efficiently coordinate drought responses.In this study, we demonstrate that HAT1, which was previously reported as a critical regulator in BR-mediated plant growth and in viral defense response, is involved in ABA regulation of drought response by suppressing the ABA biosynthesis and signaling. We found that HAT1 and its homolog HAT3 act redundantly, as the expression of both http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi), we found that HAT1 expression was reduced after ABA and osmotic stress treatment in seedlings, implying that HAT1 may be implicated in ABA and stress responses. To test the hypothesis, we examined the expression of HAT1 in different tissues and in seedlings treated with exogenous ABA or osmotic stress. Consistent with the public data, the expression level of HAT1 was highest in root, and lower in stem, leaf, and inflorescence  mutants  and was similar among Col-0, hat1, hat2, hat3 and hat1hat2 with or without ABA treatment. To analyze the function of HAT1 in osmotic stress tolerance, 4-day-old Col-0 and knockout mutants were treated with mannitol, a stress treatment commonly used to mimic osmotic stress tolerance in the laboratory. The double mutant hat1hat3 or triple mutant hat1hat2hat3 displayed less inhibition on growth in the medium containing mannitol compared with Col-0, while the single mutant hat1, hat2, hat3 and double mutant hat1hat2 showed little difference after mannitol treatment in comparison with Col-0 (HAT1-overexpressing lines (HAT1OX#11 and HAT1OX#13) showed significantly reduced ABA sensitivity and osmotic stress tolerance  or hat1hat2 showed little difference after ABA treatment in comparison with Col-0   and 10 μCi 32P ATP. After incubation at 37°C for 60 min, the reactions were stopped by adding 20 μL of 2×sodium dodecyl sulfate (SDS) buffer and boiling for 5 min. Proteins were resolved by polyacrylamide gel electrophoresis (PAGE) and phosphorylation was detected by exposing to a phosphor screen, and signals were obtained by a Typhoon 9410 phosphor imager. The in vivo phosphorylated HAT1 was examined by Phostag reagent (NARD Institute) with or without CIP treatment as described Guan et al  with approximately 1 ng probe (10000 cpm) and recombinant proteins purified from E. coli. After 30 min incubation on ice, the reactions were resolved by 5% native polyacrylamide gels with 1×TGE buffer . The assays were repeated three times with similar results.EMSA was performed using an Electrophoretic Mobility-Shift Assay (EMSA) Kit *with SYBR Green and SYPRO Ruby EMSA stains* . The level of precipitated DNA fragments was quantified by RT-qPCR using specific primer sets  GUS staining for expression patterns of HAT1. Transgenic plants expressing HAT1-Promoter::GUS at seedlings (left) and leaves of adult plants (right) were stained with 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc). GUS expression was examined in cotyledons, roots and guard cells before and after 100 μM abscisic acid (ABA) treatment for 3 h and mannitol treatment for 6 h. (B) Subcellular localizations of HAT1-GFP. Protoplasts from wild-type (WT) plants were transformed with 35S:HAT1-GFP or 35S:GFP. The signals were observed under a fluorescence microscope. GFP, green fluorescent protein. Cell images were also taken under bright field as a control. Bars, 20 μm.(TIF)Click here for additional data file.S2 FigHAT1, HAT2 and HAT3 in T-DNA insertion mutants. (B) HAT1 protein was detected by western blotting with anti-GFP antibody. Similar HAT1 protein levels in HAT1OX#11 and HAT1#13 line.(A) Reverse transcription-PCR was employed to estimate the transcription levels of (TIF)Click here for additional data file.S3 FigHAT2 and HAT3 in response to ABA and osmotic stress. 12-day-old Col-0 seedlings were transferred to liquid MS medium containing 100 μM ABA and 200 mM mannitol and then the plants were harvested at the indicated time. Actin2 was used as the internal control. Data are shown as mean ± SD of three independent experiments. (C) Growth of different genotype seedlings on 1/2 MS medium with/without 10μM ABA or 200 mM mannitol. The 4-day-old seedlings were transferred to 1/2 MS or 1/2 MS medium supplemented 10μM ABA or 200 mM mannitol for 10 days, and then the photos were taken. (D-F) Quantification of primary root length and biomass in different genotypes after ABA treatment or mannitol treatment indicated in (C). The average and SDs were from three replications, asterisks indicate significant differences compared with Col-0 under the same treatment conditions. The significant difference was analyzed by Student’s t test . Expression patterns of (TIF)Click here for additional data file.S4 Fig(A) Epidermal peels of indicated genotypes were treated with or without ABA for 2 h after stomatal pre-opening under light for 3h, and the stomatal aperture was measured by microscope. Scale bars: 10μm. (B) Stomatal apertures of different genotypes indicated in (A). Bars indicate SD calculated from three replications and at least 20 stomatals were measured for each genotype per replication. The significance of difference was analyzed by Student’s t test (**P < 0.01).(TIF)Click here for additional data file.S5 Fig(A) Phenotypes of different genotypes in response to progressive drought stress. 4-week-old plants were subjected to drought stress by withholding watering (drought) for 14 days , followed by rehydration for 5days. (B) Membrane stability status of different genotypes subjected to drought stress for 7 days and 14 days. Data is presented as a percentage of electrolyte leakage. Data are shown as mean SD of three independent experiments. The significance of difference was analyzed by Student’s t test . (C) Percentage of plants that survived the treatment mentioned in (A). Survival rate was recorded 5 days after rewatering. Bars indicate SD calculated from three replicated experiments. The significance of difference was analyzed by Student’s t test .(TIF)Click here for additional data file.S6 Fig(A) Schematic representation of a series of truncation mutations of HAT1. HD, homeodomain; LZ, leucine zipper domain. (B) BiFc assay for the interaction of SnRK2.3 with HAT1 fragments. The truncated HAT1 fragments were fused with n-YFP and co-expressed with SnRK2.3-cYFP, respectively. (C) SnRK2.3 interacts with N-terminal region of HAT1 in GST pull-down assay. GST, GST-SnRK2.3 and MBP-tagged different domains of HAT1 were used in this assay. MBP-tagged domains of HAT1 were detected by western blotting with anti-MBP antibody.(TIF)Click here for additional data file.S7 FigNicotianabenthamiana leaf epidermal cells expressing HAT3-GFP exposed to 50 μM ABA. The experiment was repeated three times with similar results and representative photos were displayed. Scale bar: 50 μm.(A) BiFC analysis of SnRK2.3 and HAT3 interactions with fusions to N- and C-terminal fragments of YFP, respectively. The constructs were expressed in tobacco leaves and the reconstitution of YFP is determined. Scale bars: 50 μm. (B) Time microscope images of (TIF)Click here for additional data file.S8 FigHAT1 (A) and SnRK2.3 (B) was tested by qRT-PCR in Col-0, HAT1OX#13 and SnRK2.3OX/HAT1OX. Data are shown as mean ± SD and Three independent experiments were done (Student’s t-test: ** P<0.01).The expression of (TIF)Click here for additional data file.S9 FigHAT1OX#13, and SnRK2.3OX/HAT1OX plants subjected to progressive drought stress. In F, data is presented as a percentage of electrolyte leakage. The average and SDs were from three biological repeats in A. The significance of difference was analyzed by Student’s t test .(A-C) Drought phenotypes (A), membrane stability status (B) and survival rates (C) of Col-0, (TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


WD repeat domain 62 (WDR62) cause microcephaly and a wide spectrum of severe brain malformations. Disruption of the mouse ortholog results in microcephaly underlain by reduced proliferation of neocortical progenitors during late neurogenesis, abnormalities in asymmetric centrosome inheritance leading to neuronal migration delays, and altered neuronal differentiation. Spindle pole localization of WDR62 and mitotic progression are defective in patient-derived fibroblasts, which, similar to mouse neocortical progenitors, transiently arrest at prometaphase. Expression of WDR62 is closely correlated with components of the chromosome passenger complex (CPC), a key regulator of mitosis. Wild type WDR62, but not disease-associated mutant forms, interacts with the CPC core enzyme Aurora kinase B and staining of CPC components at centromeres is altered in patient-derived fibroblasts. Our findings demonstrate critical and diverse functions of WDR62 in neocortical development and provide insight into the mechanisms by which its disruption leads to a plethora of structural abnormalities.Recessive mutations in  WDR621516181920WDR62 were initially discovered33343334363738Malformations of cerebral cortical development (MCD) are the outcome of disruptions in the precisely orchestrated series of events that occur during embryonic and early postnatal life and define the specification of neuronal identity and, ultimately, the execution of neuronal differentiation and connectivity both within and outside the cerebral cortex. MCD are neurodevelopmental disorders invariably causing developmental delay and were initially classified according to the phase at which disruptions of normal developmental events were thought to occur, impacting for example neural progenitor proliferation or apoptosis, neuronal migration, or cortical organization13461213WDR62. Our studies demonstrated that disruption of WDR62 impairs proliferation of neocortical progenitors during late neurogenesis underlain by abnormalities in asymmetric centrosome inheritance causing microcephaly in mice, and impairs mitotic cycle progression in patient-derived fibroblasts, which, similar to mouse neocortical progenitors, transiently arrest at prometaphase. Moreover, transcriptomic analyses identified genes with expression profiles significantly correlating with WDR62 across different human brain regions and developmental periods, including those encoding components of the Chromosome Passenger Complex (CPC), a major regulator of mitosisWDR62 mutations causing microcephaly.We investigated WDR62 function using a mouse model and dermal fibroblasts from affected (homozygote) and unaffected (heterozygote) members of a family carrying a novel truncating mutation in Wdr62 mRNA is expressed in proliferating neural progenitors in the ventricular and subventricular zones  of the dorsal telencephalon  as early as E11.5, in ventral  progenitors that give rise to striatal neurons and cortical interneurons, and in proliferating granule cell precursors in the external granular layer (EGL) of the cerebellum at early postnatal stages . In the developing human brain, WDR62 mRNA is enriched in the VZ, weakly expressed in the intermediate zone and cortical plate, whereas in adult human brain, it is detected throughout the neocortical layers (l stages 15. In adl layers .Wdr62 from trapped ES cells (Wdr62Gt(CH0428)Wtsi) obtained from the Welcome Trust Sanger Institute (www.informatics.jax.org). The insertion site of the pGTOlxr gene-trap vector is between exons 21 and 22 of the Wdr62 locus  express a fusion protein consisting of amino acids 1–870 (of 1524) of WDR62 fused to the β-geo reporter, detected by x-gal staining, in accordance to the endogenous pattern  and offspring number, likely reflecting defects in the germline. Several MCPH2- associated mutations identified in patients also lead to C-terminal truncations of variable length  but no difference in the medial to lateral axis , with the exception of P3 brains  , measured at different levels along the rostral to caudal axis at P3   or of cerebral cortical  weight was significantly reduced, as was the length of the A-P axis   compared with wild type    . In addir brain) . In adul, n = 4) . By appl = 0.01) . Nuclear5 and 6) , suggestisrupted , with deisrupted . These disrupted , were leisrupted . At lateenlarged . These oWdr621-21/1-21 brain at E11.5, E13.5 and E15.5 following a 30-minute pulse (Wdr621-21/1-21 embryos compared with wild type at E15.5 (+Ki67−/CldU+) during the 24-hour chase period by double labeling with antibodies specific for CldU and Ki67, which labels all dividing cells. The 24-hour cell cycle exit index was not significantly different in embryos injected at E13.5 and harvested at E14.5 (Wdr621-21/1-21embryos injected at E16.5 and harvested at E17.5 compared with wild type (+Ki67−)Wdr621-21/1-21vs. wild type neocortex at E17.5  fluorescent centrioles; about 10% of centrosomes possessed only green fluorescent centrioles, whereas less than 5% were solely red fluorescent , in agreement with previous observationsWdr621-21/1-21compared with wild type (65% vs. 40%), whereas, the percentage of those retaining the old mother centriole  was lower (Wdr621-21/1-21brains had fewer centrosomes containing green only centrioles (Wdr621-21/1-21neocortex more cells inherited the new mother centriole (green), which is typically inherited by the newly differentiating neurons migrating away from the VZ/SVZ. Accordingly, the distribution of cells containing new mother centrioles only (i.e. green fluorescent) within the developing neocortex , was different in Wdr621-21/1-21compared with wild type neocortex, with the former harboring significantly more in the proliferating zones and fewer in the migration zone and the cortical plate, suggesting abnormal migration, and possibly differentiation, of Wdr621-21/1-21neurons inheriting the new mother centriole.In non-neuronal cells, and dependent on cell line examined, WDR62 is detected at the spindle poles during at least some phases of mitosis151722273335as lower . The oppntrioles  and a hintrioles . These dWdr621-21/1-21 neocortex  was similar in wild type and Wdr621-21/1-21neocortex , caused by a 4 bp deletion in exon 23, resulting in a premature stop codon at position 1067; the mutation was confirmed to be homozygous in both affected subjects, and heterozygous in both parents, using Sanger sequencing  compared with other cells, including fibroblasts . These results suggested that the predominant consequence of MCPH2-associated mutations is the disruption of the dynamic shuttling and subcellular localization of WDR62 during mitosis. These observations further support the conclusion that the failure to detect WDR62 expression in the spindle poles of patient-derived WDR62exon23 homozygous fibroblasts indeed reflects defective shuttling of the mutant protein.To evaluate the functional consequences of additional roblasts . These ablasts 15. On veryWDR62 across developmental periods  and different brain regions  and mitotic cell cycle (Benjamini adjusted P = 9.56 × 10−40) proteins. A group of 90 transcripts highly correlated  with WDR62  (WDR62exon23fibroblasts during mitosis (data not shown). Because depletion of microtubule-associated proteins is known to affect the level of CPC accumulation at centromeresW224S and WDR62exon23, two MCPH2-associated mutant forms  and aurb. We employed the GAL4/UAS system, using Prospero-GAL4 to drive expression of Wdr62 and aurb RNAi (Wdr62-IR and aurb-IR) in neuroblasts  and their newly born progeny (ganglion mother cells), which constitute the majority of cells in the developing larval brainWdr62-IR resulted in markedly reduced brain size of 3rd instar larvae as compared with controls, confirming an evolutionarily conserved role for WDR62; in contrast, aurb-IR had no obvious effect on brain size. Combined Wdr62-IR and aurb-IR, however, resulted in formation of clusters of abnormally enlarged neuroblasts in 40% of brains analyzed , identified by expression of the molecular marker Miranda; the more such clusters had developed in a brain, the smaller its size   was enrivalue 1) , or encovalue 1) . These fvalue 1) 66 as welvalue 1) . Furthervalue 1) . The dynnt forms , suggestits size . These oWDR62 in two affected siblings with microcephaly and severe brain malformations, and characterize its functional impact using patient-derived fibroblasts. We establish a critical role of WDR62 in proliferation of late neocortical progenitor cells and brain size and show that its disruption causes abnormalities in asymmetric centrosome inheritance leading to neuronal migration delays, and altered neuronal differentiation. We show that mitotic progression is defective in patient-derived fibroblasts, which, similar to mouse neocortical progenitors, transiently arrest at prometaphase. We also find that expression of WDR62 closely correlates to core components of the CPC, a major regulator of mitosis. We demonstrate that staining of CPC components at centromeres is altered in patient-derived fibroblasts and that, at least upon overexpression, mutant forms of WDR62 disrupt interactions with AURKB, the CPC enzymatic core. These findings highlight complex roles of WDR62 in mitotic cycle regulation, age-related progenitor maintenance, neuronal migration and differentiation, and may help explain the wide spectrum of structural brain abnormalities that uniquely characterize individuals with WDR62-associated microcephaly.In this study, we examine the effects of WDR62 disruption on neocortical development using a newly generated mouse model, identify a novel homozygous mutation in Wdr621-21/1-21mice harbor smaller brains, with significantly shorter anterior-to-posterior neocortical axis length and fewer cells overall, reflecting decreased proliferation and decreased cell cycle exit of late progenitors and increased apoptosis. WDR62 dysfunction further impacts mitotic progression, as evidenced by enrichment of cells at prometaphase in murine mitotic progenitors in the VS and in dividing fibroblasts from individuals with MCPH2. In the murine neocortex, WDR62 loss disrupts asymmetric centrosome inheritance, a mechanism that has been previously linked to progenitor maintenanceWdr62 ortholog also plays a role in maintaining centrosome asymmetry in neuroblasts and is required for microtubule stabilization and maintenance of microtubule organizing center activity on the apical interphase centrosomeAs in humans, disruption of WDR62 in mouse and Drosophila leads to microcephaly, highlighting its evolutionarily conserved function. Wdr621-21/1-21mouse partially confirm those from the Wdr621-14/1-14 mouse in which Wdr62 is disrupted after exon 14Wdr621-14/1-14MEFsWdr621-21/1-21 neocortical progenitors and patient-derived fibroblasts also transiently arrest in prometaphase. While Wdr621-14/1-14MEFs commonly display multipolar spindles, extra centrosomes and lagging chromosomesWDR62exon23homozygous fibroblasts harbor extra centrioles only very rarely Wtsi) obtained from the Welcome Trust Sanger Institute (www.informatics.jax.org) and maintained in accordance with the National Institutes of Health guidelines and approval of the Yale University Institutional Animal Care and Use Committee.in situ hybridization or Nissl staining. Brains of E18.5 embryos, neonatal and postnatal mice were extracted after transcardiac perfusion with cold PBS followed by 4% PFA.Embryonic brains (except E18.5) were extracted and immersed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PBS) for 24 hours and up to several days, and processed, respectively, for immunohistochemistry and Brains were harvested at the indicated stages and fixed as described above. Measurements (in mm) were obtained along the anterior-most to posterior part of the forebrain (A-P axis length) and at the widest part of the forebrain (M-L axis length), as indicated in Brains of 9-week-old mice were harvested and post-fixed by immersion in 4% PFA for 6–8 weeks. The cerebral cortex and the hippocampus were dissected and total cell numbers were estimated using the isotropic fractionator as previously describedBrains at the indicated stages were fixed, extracted, and sectioned with a sledge cryomicrotome . Nissl staining was performed on serial sections by standard procedures. Embryonic brain sections were stained for β-galactosidase activity in a solution containing 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 20 mM magnesium chloride, 0.1% Triton X-100, and 0.37 mg/ml X-gal.Pregnant females were injected intraperitoneally with a solution of 5-bromo-2′-deoxyuridine  at 20 μg/g of body weight and/or with 5-chloro-2′-deoxyuridine  at 42.5 μg/g of body weight. Brains were harvested and processed for immunohistochemistry or immunofluorescence with antibodies specific to BrdU and/or CldU.Brain sections (36 μm thickness) were obtained with a cryomicrotome (Leica), air-dried, post-fixed in 4% PFA for 15 min, washed with PBS, incubated with Proteinase K (1 μg/ml) in Proteinase K buffer  at 37 °C for 30 min, washed with PBS and processed using the Millipore S7100 apoptosis detection kit (Fisher Scientific) following the manufacturer’s instructions.in situ hybridization as described previouslyPax6Tbr2Cux288Er81 , Notch1, reelin, Scip , Rorb ], or generated using oligonucleotide pairs as indicated below  and sectioned with a cryomicrotome (Leica). Human tissue was obtained from several sources, including the Human Fetal Tissue Repository at the Albert Einstein College of Medicine , fixed by immersion in 4% PFA and sectioned. Sections were processed for in situ probes as shown in Oligonucleotides used to generate the For immunofluorescence staining of sections at prenatal stages, brains were harvested and fixed as described above. The brains were cryoprotected by sequential immersion in 15% and 30% sucrose in PBS, flash frozen into embedding medium  and sectioned using a cryostat (Leica). Sections (12 μm) were air-dried, post-fixed in 4% PFA for 10 min and washed with PBS 3 times for 5 min each, blocked in a solution containing 5% normal donkey serum , 1% bovine serum albumin, 0.1% glycine, 0.1% L-lysine supplemented with 0.3% Triton X-100 for 1 hour and then incubated in the above solution with the appropriate primary antibody overnight at 4 °C. Sections were washed 3 times in PBS for 10 min each and incubated with secondary antibodies [Alexa-Fluor conjugated ] in blocking solution without detergent for 2 hours at room temperature. For immunofluorescence staining at postnatal stages, brains were harvested following transcardiac perfusion as described above, post-fixed in 4% PFA overnight and cryoprotected in 30% sucrose in PBS for several hours. Sections (50 μm) were obtained on a cryomicrotome (Leica) and processed free-floating. After washing 2 times in PBS, 5 min each, sections were incubated in blocking solution (same as above) with 0.9% Triton X-100 for 1–2 hours at room temperature, followed by incubation with the appropriate primary antibodies in the same blocking solution for 72 hours at 4 °C. Sections were washed 3 times with PBS, 15 min each and incubated with the appropriate secondary antibodies in blocking solution without detergent for 2 hours at room temperature.Human fibroblasts, iPSCs, murine embryonic cortical neurons and HeLa cells were fixed with cold 4% PFA for 15 min at room temperature, washed 3 times with PBS, incubated in blocking solution (same as above) for 1 hour and then in primary antibody in blocking solution for 1 hour at room temperature. After 3 washes with PBS, secondary antibodies were applied in blocking solution without detergent for 1 hour at room temperature. Samples were mounted with Vectashield containing DAPI (Vector Laboratories), and imaged with a Zeiss LSM or a Leica TCS SP2 laser scanning confocal microscopy system.WDR62 ; BrdU , CldU , Ki67 , SATB2 , CTIP2 , TBR1 , WDR62 , gamma-tubulin , ASPM , AIM-1 , CEP63 , CEP135 , PH3 , CDK5RAP2 , BIRC5/survivin , CREST ; myc ; V5 ; V5 .In utero electroporation was performed using standard proceduresAll surgical procedures were performed using sterile conditions and in accordance with IACUC approved protocols. Cortical leaves from E15.5 embryos were dissected in cold Hanks’ Balanced Salt Solution (HBSS) supplemented with 0.5% D-glucose and 25 mM HEPES (HBSS+). Minced cortical tissue samples were dissociated using Papain-Protease-DNAse I (PDD) solution  and incubated at 37 °C for 30–40 min. Samples were subjected to centrifugation at 500 g for 4 min and pellets were resuspended in 1 ml of Neurobasal medium supplemented with 1 mM Sodium Pyruvate, 2 mM Glutamax, Pen/Strep and B-27 Supplement and filtered through a 40-micron mesh filter. Cells were plated on glass coverslips coated with laminin and poly-ornithine and cultured in Neurobasal medium as above.2 Petri dishes, in square areas marked by perpendicular lines made with scalpel blades. The fragments were allowed to air dry so as the dermis side of specimens adhered to the culture dish. The fragments were then cultured in DMEM with 10% FBS, 1% (1x) L-glutamine, 1% (1x) Penicillin-Streptomycin at 37 °C. The fibroblasts typically grew within 7–9 days.A 4-mm skin punch biopsy was taken under local anesthesia from the umbilical area of NG1406-1, NG1406-2, and their parents  using standard procedures93Human fibroblasts and HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum. To synchronize the fibroblast cultures, cells were rinsed with PBS and grown in serum-free medium for 18 hours, then seeded in gelatin-coated chamber slides and released into the cell cycle for 36 hours by addition of serum prior to fixation with 4% PFA and analysis. For arrest in prometaphase, asynchronous fibroblast cultures were treated with 0.2 μg/ml nocodazole (Sigma) for 14 hours, then fixed for analysis. Primary fibroblasts synchronized in prometaphase were washed and released into growth medium for 1 hour, fixed, and analyzed for progression to metaphase.5) were nucleofected with 3 micrograms of Kaede-CETN1 plasmidFibroblasts were harvested by centrifugation and processed for transfection using a Nucleofector 2b device  and a kit for Human Dermal Fibroblasts  according to the manufacturer’s instructions. Cells . 36 hours after transfection cells were treated with 0.2 μg/ml nocodazole (Sigma) for 12 hours, then were lysed using a Triton-X100 based buffer containing protease and phosphatase inhibitor cocktails . Clarified supernatants were used for immunoprecipitation using Dynabeads (Life Technologies) according to the supplier’s instructions. Immunoprecipitates were analyzed by Western analysis using standard protocols. In brief, lysates were separated on gradient SDS-PAGE gels  and transferred to nitrocellulose or PVDF membranes. After blocking with 5% milk, the membranes were incubated first with rabbit anti-tag antibodies and then with a secondary HRP-conjugated anti-rabbit IgG (Jackson Immunoresearch Labs). Signals were detected with chemiluminescence reagent (BioRad).t-test or Mann-Whitney U test with a significance level of at least P < 0.05 . Values designate mean; error bars represent s.e.m.All data obtained were analyzed using two-tailed Student’s + cells or labeled centrosomes were counted in each bin as indicated in the respective graphs.To quantify the distribution of neurons, anatomically matched sections were selected. The neocortex of P3 (SATB2 immunofluorescence) or E18.5 brains (Kaede-CETN1 expression) was subdivided radially from the pia surface to the upper edge of the white matter, into 10 or 3 equally sized bins, respectively. SATB2http://imagej.nih.gov/ij). All images were processed with Image J and/or Adobe Photoshop.Visualization of stained brain sections or cells was performed using a Stemi stereomicroscope or an AxioImager (Zeiss) fitted with an AxioCam MRc5 digital camera, or using a TCS SP5 confocal microscope (Leica). Images were captured using the AxioVision (Zeiss) or the integrated LAC (Leica) software. Confocal images were assembled using LAS AF lite  or Image J . Gateway BP and LR reactions were performed using BP and LR clonase, respectively, according to the vendor’s instructions. PCR reactions to amplify genes and/or gene fragments were performed using the high-fidelity Accuprime Pfx DNA polymerase (Life Sciences) while all mutagenesis reactions were performed using the high-fidelity Phusion DNA polymerase (New England Biolabs). Gateway B1/B2 PCR reactions were performed using primers below, in order to amplify the cDNA of interest. To design primers, we used the QuickChange Primer Design website . B1/B2 PCR products containing the cDNA of the desired gene were purified using the PCR purification kit (Qiagen) and were recombined using BP clonase into the shuttle plasmid pDONR-zeo (Life Sciences). This yielded so-called cDNA ENTRY clones. All genes were fully sequenced at this stage. When needed, site-directed PCR mutagenesis was performed on cDNAs ENTRY clones, then fully sequenced. Once the sequence of any ENTRY clone was confirmed, the respective plasmid DNA was recombined using LR clonase into a common, CMV promoter-driven expression vector, pCDNA-DEST40 (Life Sciences), which carries a V5-HIS tandem tag at the 3′ end of the Gateway cassette. All B2 primers depicted below have no STOP codon, allowing tagging of all genes with the V5-HIS tandem tags present in pCDNA-DEST40.WDR62 cDNA sequence via standard PCR mutagenesis using Phusion polymerase.pCAN3Myc-WDR62 (encoding the CS5 transcript of WDR62) was a gift from A. Aronheim WDR62 cDNA. The same mutagenesis protocol and the respective primers (see below) were used to introduce the W224S missense mutation. To generate the shorter, truncated versions of WDR62 mutations were introduced in exon 23 (Asp955AlafsX112), intron 23 (S956CfsX38), and exon 31 (V1402GfsX12), using Gateway with reverse (B2) primers targeting the respective regions. Since the GACA deletion in exon 23 causes a frameshift that adds 112 amino acids until the next “stop” codon, we used PCR insertional mutagenesis to introduce the 112-amino acid tail to the previously made exon 23 deletion construct. The above PCR product obtained with the W62xMF/MR primer pair was purified and used for mutagenesis to insert the 112-amino acid tail “in-frame” at the end of the construct previously generated with the B2nsEX23 W primer. In all constructs the STOP codons at the end of the ORF were deleted to allow 3′ tagging of the respective cloned genes with the V5-His tandem peptide tags present within the DEST40 plasmid.Four mutations described in patients with MCPH2 were introduced in the wild type A full-length cDNA encoding Aurora Kinase B was purchased from Addgene (plasmid 23682: pDONR223-AURKB). The gene was recombined via LR reactions into the pcDNADEST40 Gateway expression vector. In addition, the V5-His tag was removed and replaced with a 3x Myc tag.All constructs generated were confirmed by Sanger sequencing.WDR62 full length gene:Gateway oligonucleotides used to subclone the B1-W62: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCACCATGGCGGCCGTAGGGTCCGGAGGCTATGB2nsW62: GGGGACCACTTTGTACAAGAAAGCTGGGTCCCAGTGCCCCCGTGCCTTCCTCCGCACOligonucleotides used to insert the 194 bp fragment into the original clone:F: GTGCTGGAGAAGTGGATCAACCTGAAGR: GTCGAAGGTCAGTGCCACTGTATCTGGGateway oligonucleotides used to subclone WDR62 truncated proteins:B2nsEX31W: GGGGACCACTTTGTACAAGAAAGCTGGGTCCCAGGGCTCACTCCAGCGGGCAGGGCTGCCCTGTAACAB2nsEX23W: GGGGACCACTTTGTACAAGAAAGCTGGGTCGCTGTCTGTCCCTGTGACTGTCACTTCOligonucleotides used to perform the W224S mutagenesisW224SF: CACTGTTGGGAACCGCCATGTGAGGTTCTCGTTCTTGGAAGTCTCCACTGAGACAAAGW224SR: CTTTGTCTCAGTGGAGACTTCCAAGAACGAGAACCTCACATGGCGGTTCCCAACAGTGOligonucleotides used to add the “tail” of 112 amino acids in the exon 23 constructW62 × 22MF: TACTCTCTGGAGGCAGAAGTGACAGTCACAGAGACAGCCAGTATTGCAGGAAGGAGGTGW62 × 22MRns: AACTTTGTACAAGAAAGCTGGGTCGTGTCTCAAAGTGGTGGCGGAGGAACTTCTThe study protocol was approved by the Yale Human Investigation Committee (HIC) (IRB #9406007680). Institutional review board approvals for genetic and MRI studies, along with written informed consent from all study subjects, were obtained by the referring physicians at the participating institutions. Human fetal brain specimens at 19 weeks of gestation were obtained from the Human Fetal Tissue Repository at the Albert Einstein College of Medicine (CCI number 1993–042). Postmortem human brain specimens were obtained from tissue collections at the Department of Neuroscience at Yale School of Medicine.nd centile; height: 116 cm, 3rd–10th centile; weight: 20 kg; 3rd–10th centile). He never developed the ability to speak. Large auricles were noted. The index case’s 6-year-old brother (NG1406-2) displayed similar symptoms. He was brought to medical attention as soon as he was born with microcephaly . He developed head control at 3 months and ability to sit without support at 7 months of age. Global developmental delay was detected with the DENVER developmental screening test, performed at 18 months. At age 2 years, head circumference was 41 cm (<2nd centile), height was 83 cm (10th–25th centile) and weight was 10 kg (<3rd centile). General examination was remarkable for global developmental delay, no ability to walk, hypoplastic forehead and large auricles. At age 3 1/2, head circumference was 46.5 cm (<2nd centile), height was 129 cm (10th–25th centile) and weight was 22 kg (<3rd centile). Like his brother, he exhibited microcephaly and no ability to speak fluently. Their routine blood studies, including neurometabolic assays, complete metabolic panel, complete blood count, and thyroid function tests were all within normal limits. Whole exome sequencing of the younger affected brother identified a novel homozygous frameshift mutation, D955AfsX112, caused by a 4 bp deletion in exon 23 of WDR62, resulting in a premature stop codon at position 1067; the mutation was confirmed to be homozygous in both affected subjects, and heterozygous in both parents, using Sanger sequencing. It was not observed in more than 1,800 Turkish control chromosomes, or in the ESP (exome sequencing project) and 1,000 genome databases.The index case (NG1406-1) is a 13-year-old full term male who is the product of a second-cousin marriage. He presented for medical attention at 20 months of age owning to global developmental delay and was found on clinical examination to have microcephaly and dysmorphic facies. Neuroimaging studies identified an array of developmental abnormalities including diffuse pachygyria, thickened cortex and hypoplasia of the corpus callosum and three-dimensional computed tomography showed metopic synostosis. At 8 years of age, he appeared microcephalic  according to the manufacturer’s protocol along with modifications previously described15Analysis of the sequencing data was performed according to the previously described bioinformatics pipeline devised by our research teamThe mutation was evaluated by Sanger sequencing using standard protocols. Amplicons were cycle sequenced on ABI 9800 Fast Thermo cyclers , and post cycle sequencing clean-up was carried out with the CleanSEQ System . The amplicons were analyzed on a 3730 Å~L DNA Analyzer (Applied Biosystems Inc.).How to cite this article: Sgourdou, P. et al. Disruptions in asymmetric centrosome inheritance and WDR62-Aurora kinase B interactions in primary microcephaly. Sci. Rep.7, 43708; doi: 10.1038/srep43708 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Resistance-associated variants have been related to treatment failure of hepatitis C virus (HCV) therapy with direct-acting antiviral drugs. The aim of our study was to analyze the prevalence of clinically relevant resistance-associated variants within NS3 in patients infected with HCV genotype 1a (GT1a) in Spain.http://hcv.geno2pheno.org/).We performed a cross-sectional study on 2568 patients from 115 hospitals throughout Spain (2014–2015). The viral NS3 protease gene was amplified by nested polymerase chain reaction and sequenced by Sanger sequencing using an ABI PRISM 377 DNA sequencer. Additionally, clade information for genotype 1a was obtained by using the software geno2pheno /HCV-coinfected patients. Q80K was the main RAV found in our patients (11.1%) and the rest of the resistance-associated variants had a lower frequency, including S122G (6.23%), T54S (3.47%), V55A (2.61%), and V55I (2.15%), which were among the most frequent after Q80K. Overall, 286 samples had the Q80K polymorphism (11.1%) and 614 (23.9%) were GT1a clade I. HIV/HCV-coinfected patients had a higher frequency of Q80K and GT1a clade I than HCV-monoinfected patients . Both the prevalence of Q80K and GT1a clade I were not uniform throughout the country (p<0.001), which ranged from 7.3%-22.2% and 15.7%-42.5%, respectively. The frequency of the Q80K polymorphism was far higher in patients infected with GT1a clade I than in patients infected with GT1a clade II .The prevalence of most resistance-associated variants in NS3 was low in patients infected with HCV GT1a in Spain, except for Q80K (11.1%), which was also notably higher in HIV/HCV-coinfected patients. The vast majority of Q80K polymorphisms were detected in GT1a clade I. Hepatitis C virus (HCV) therapy has changed rapidly with new direct-acting antiviral drugs (DAAs), particularly for HCV genotype 1, achieving high rates of sustained virologic response . Thus, pin-vitro phenotypic assays  and Europe [clade II]) with and without Q80K, respectively ,25. CladThis study has relevant limitations, some of which have been discussed above: (i) the number of patients found exhibiting baseline RAVs (not including Q80K) was quite low and does not allow us to draw strong conclusions for some RAVs; (ii) the association of RAVs with SVR data were not assessed in this study, but the clinically relevant RAVs within NS3 were extrapolated from a recent review by Lontok et al. [In conclusion, the prevalence of NS3 RAVs was low in patients infected with HCV GT1a in Spain, except for Q80K (11%), which was higher in HIV/HCV-coinfected patients. The vast majority of the instances of the Q80K polymorphism was detected in GT1a clade I, but it was also detected in GT1a clade II.S1 Table(DOCX)Click here for additional data file.


Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function of each amino acid residue involved in various stress conditions including exposure to different DNA damage-inducing reagents, different growth temperatures, and other chemicals. H2B N- and H2A C-termini were critical for maintaining nucleosome function and mutations in these regions led to pleiotropic phenotypes. Additionally, two screens were performed using this library, monitoring heterochromatin gene silencing and genome stability, to identify residues that could compromise normal function when mutated. Many distinctive regions within the nucleosome were revealed. Furthermore, we used the barcode sequencing (bar-seq) method to profile the mutant composition of many libraries in one high-throughput sequencing experiment, greatly reducing the labor and increasing the capacity. This study not only demonstrates the applications of the versatile histone library, but also reveals many previously unknown functions of histone H2A and H2B. In eukaryotes, genomic DNA is compacted into chromatin to fit into the nucleus. As the functional unit of chromatin, a nucleosome consists of histone octamers—including two copies each of the four core histones H2A, H2B, H3, and H4—and ∼147 bp DNA  and SHY15 (a derivative of W303) to screen the mutants that affect heterochromatin silencing and genome stability, respectively. Strains in the BY-H2ML1 library were used for methyl methanesulfonate (MMS) assay by bar-seq or SD. Genotype of all strains are listed in Supplemental Material, Table S1 in File S1.Strains in the BY-H2ML1&2 library were used for high-throughput phenotype analysis under different stress conditions with serial dilution (SD). Linearized plasmids of H2ML1 by Synthetic complete (SC) medium contained 0.17% (w/v) yeast nitrogen base without amino acids and ammonium sulfate , 0.5% (w/v) ammonium sulfate, 2% (w/v) glucose, and 2% (w/v) bacto-agar, supplemented with 0.2% (w/v) amino acids drop-out powder. The concentration of 5-FOA in synthetic media was 1 g/L. Drug-containing media were prepared by supplementing YPD with 200 mM hydroxyurea (HU), 0.03% (v/v) MMS, 10 μg/ml benomyl, 8 μg/ml camptothecin (CPT), 2 μg/ml nocodazole, 25 nM rapamycin, or 0.3% (v/v) acetic acid, respectively.SUP11 and HIS3 markers  was derived from SBY8054, which contains an artificial chromosome III fragment with MET15 and ADE2, within the rDNA repeat and the subtelomeric region on chromosome V, respectively  for 1 wk at 30° before we scored the colony color (The reporter strain (JDY187) was derived from YNB12, containing two makers, 2O twice before inoculation into 50 ml YPD for overnight growth at 30°. The cell density of the overnight cultures was measured and 2.5 × 108 cells from both cultures were harvested for genomic DNA preparation.Viable H2A/H2B mutants were pooled together and stored in 15% glycerol at −80°. For the MMS assay, cells of the H2A/H2B pool were inoculated in 50 ml of YPD for 4 hr at 30°. Next, 10 ml of cell culture was transferred into two new tubes: one with MMS  and the other without. The cells were cultured at 30° for 2 hr, precipitated by centrifugation, and washed with ddHFile S1)  and integrated at the endogenous loci, respectively, a series of phenotypic analyses was performed. Four classes of growth conditions were evaluated, including: (i) stresses that lead to DNA damage , (ii) microtubule destabilization (benomyl and nocodazole), (iii) temperature stresses (16 and 39°), and (iv) others (rapamycin and acetic acid) (Table S2 in File S1). The degree of sensitivity of H2A/H2B mutants under each condition was arbitrarily classified (from 1 to 4), representing increased sensitivity. The complete phenotype data are listed in Table S3.After constructing the systematic yeast strain library in which each cell contains two copies of the same mutant histone, under the control of their native promoters (File S1). For example, we identified 109 mutants covering 76 amino acid residues that showed sensitivity to HU to different extents. When compared to previous screens (File S1). Additionally, it is notable that two mutants of native alanine residues, H2A A61S and A70S (a residue commonly omitted from most mutagenesis studies), were found to be sensitive not only to DNA damage reagents, but also to drugs that disrupt microtubules (Table S3). Furthermore, besides testing if the yeast strains could survive at higher temperature (39°), we screened the mutants that were sensitive to lower temperature (16°). These mutants could potentially be applied to study the mechanisms of adaptation to low temperature, which remain largely unknown , suggesting that histone H2A and H2B are important in maintaining normal nucleosome function. The positions of the mutated residues with phenotypes were mapped to the nucleosome structure and represented by different colors ( screens . SimilarFile S1). Furthermore, among the mutants with phenotypes, >60% of mutants showed at least two classes of phenotype . Deletion of residue 33–36 moves amino acids 29–32 to the same position, where they might fulfill a similar function. From the nucleosome structure, R36, a conserved residue from yeast to human, interacts with the minor groove of surrounding DNA and the H2B N-terminal tail protrudes out of the nucleosome between two strands of DNA . The positively-charged K and R within the XKXR motif at this location might mediate interactions between histone H2B and DNA, thereby stabilizing the nucleosome structure. In support of this hypothesis, point mutations at either residue (K34E of R36E) also showed DNA-related phenotypes (Table S3). Since these residues were previously mutated only to alanine, none of these residues were identified in previous studies , giving a much more comprehensive view of residues involved in genome stability in all four core histones (Our previous study indicated that many residues on histone H3 and H4 are required to maintain a stable genome . The affhistones .File S1) (File S1), which were mainly contributed from the N-terminal deletion mutants of these two histones (File S1) (File S1). As expected, the chimeric H4 significantly rescued the chromosome loss phenotype. We further showed that the positively-charged lysine residues in the interaction region are required since mutations of these residues led to a failure to rescue the phenotype . (4) Finally, there are a few mutants, such as H3 K115A and H2A T77D, which locate approximately to the DNA–histone interface, which were also identified. These mutants might the loosen interaction between histones and DNA, leading to unstable nucleosomes.Several nucleosome regions were revealed by this analysis. (1) Residues near the DNA entry and exit sites, such as mutations in the H3 α N helix and the nearby H2A C-terminal , are impADE2 and MET15 within the rDNA repeat was used as the reporter strain , including two that also showed an LRS phenotype (Table S5 in File S1). On the other hand, seven long deletion mutants of the H2B N-terminal (at least 16 amino acids deleted) displayed obviously increased telomere silencing (ITS), while another deletion mutant (H2B del29-36) showed an obvious LTS phenotype (File S1). The dense thicket of silencing phenotypes mapping in a complex manner reveals not only the different behaviors of the two histone tails, but also the existence of distinct roles for silencing regulation within subregions of the H2B N-terminal tail.Mapping the identified residues onto nucleosome structure revealed the existence of shared and distinct regions between loss of telomere (LTS) and loss of rDNA silencing (LRS) phenotypes . StrikinFile S1 depict the sequencing reads from treated samples relative to the untreated controls. Mutants with a log2 ratio below −1 were defined as MMS-sensitive candidates. The two repeats predicted 56 or 50 candidates, respectively, and the 45 overlapping candidates  were used for further analysis.The spotting assay commonly used to identify the sensitivity of a strain under certain stress conditions is simple and straightforward , allowinNext, we compared the bar-seq result with that from SD assays performed on each individual mutant. The N-terminal tails of H3 and H4 are known hot spots of PTM and play many critical biological roles (Table S3), suggesting distinct mechanisms in maintaining genome integrity for the two tails. Although the H2A C-terminal tail is reportedly critical for docking Sgo1 and thereby maintaining regular chromosome segregation (Table S3). Taking the position of the HBR domain and mutant phenotypes together, we propose that the disruption of the interactions between basic residues in the HBR domain and DNA affected nucleosome functions by altering their structural stability. Consistent with this, deletion of the HBR domain causes increased nucleosome accessibility and decreased histone occupancy in the genome , indicating important roles of the residues on the H2A C-terminus in the repression of silent chromatin. At the same time, S121D and S121A also showed LTS and LRS phenotypes, respectively. Although phosphorylation of serine 121 is critical for chromosome segregation (File S1). Among them, four mutants had the truncated H2B N-terminal tail in which part or all of the HBR domain was also deleted, such as del1-32 and del5-36. The opposite effects on silencing for overlapping deletions strongly suggests the existence of antagonistic mechanisms within distinct subregions of the H2B N-terminal tail. Based on the literature, six lysine residues  are acetylated and maintain hypoacetylation in silencing mating loci and telomeric regions (File S1), complicating mechanistic interpretation.Second, mutants of the two tails showed dramatically different patterns of telomere silencing . DeletioTable S3 and Table S4 in File S1). In the nearby C-terminal motif, there is the earliest mark of the double strand break: γH2AX (Third, from the results of DNA damage stresses, the residues from 128 to 131 on H2A were sensitive to MMS but resistant to HU, while no regions in H2B showed this type of pattern (These versatile next-generation histone mutant libraries promise to uncover many additional phenotypes associated with DNA packaging, transcription, replication, and repair, and are being made publicly available.www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300252/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.