Xist has Xist-specific repeat elements A-F which are conserved among eutherian mammals, underscoring their functional importance. Here we report that Xist RNA repeat E, a conserved Xist repeat element in the Xist exon 7, interacts with ASH2L and contributes to maintenance of escape gene expression level on the inactive X-chromosome (Xi) during XCI. The Xist repeat E-deletion mutant female ES cells show the depletion of ASH2L from the Xi upon differentiation. Furthermore, a subset of escape genes exhibits unexpectedly higher expression in the repeat E mutant cells than the cells expressing wildtype Xist during X-inactivation, whereas the silencing of X-linked non-escape genes is not affected. We discuss the implications of these results to understand the role of ASH2L and Xist repeat E for histone modifications and escape gene regulation during random X-chromosome inactivation.Long non-coding RNA Xist plays a crucial role in establishing and maintaining X-chromosome inactivation (XCI) which is a paradigm of long non-coding RNA-mediated gene regulation.  Xist long noncoding RNA induces X chromosome inactivation by recruiting multiple chromatin modifying enzymes to the inactive X-chromosome. Among such chromatin modifying enzymes, ASH2L is unique since it is a component of the MLL/SET complexes, which are a histone lysine 4 methyltransferase for transcriptional activation. Among the novel results in our paper, we found that ASH2L binds to the repeat E of Xist RNA. Using the Xist repeat E deletion mutant cells, we discovered that the repeat E of Xist RNA is dispensable for X-linked gene silencing, but is required for maintaining proper level of escape gene expression. This is the first report indicating that Xist RNA engages not only in X-linked gene silencing but also in escape gene regulation. To equalize the imbalance of X-linked gene dosage between genders, one of the two X chromosomes in female mammals is converted to the transcriptionally inactive X-chromosome (Xi) at the early phase of development whereby the majority of X-linked genes on the Xi are silenced –3. In miOnce random XCI is established in epiblast lineage, the Xi is inherited throughout successive cell divisions to continue balancing X-linked gene dosage between males and females. While the majority of X-linked genes are subjected to silencing by XCI, a subset of genes called escape genes (approximately 3% in mice) is partially transcribed on the Xi ,7. TransXic) required for XCI harbors a number of non-protein-coding (non-coding) genes such as Xist and its antisense Tsix . H3. H3Xist ntiation . AlthougXist repeat E deletion resulted in increased free ASH2L in the nucleus, the increased amount of ASH2L might disturb the MLL/SET complexes' activity. Xist RNA might act as a molecular sponge of ASH2L to regulate the MLL/SET complexes' activity. A similar sponge function of noncoding gene Jpx, a positive regulator for Xist, has been reported [Jpx expression is essential for Xist upregulation at the onset of random XCI through eviction of Xist repressor CTCF by its binding affinity with CTCF.Surprisingly, the depletion of ASH2L from the Xi by the repeat E deletion resulted in transcriptional upregulation of a subset of escape genes we tested without affecting stable silencing of non-escape genes during XCI. We initially hypothesized that escape gene expression on the Xi is repressed due to the depletion of ASH2L from the Xi, since ASH2L is a common component of the MLL/SET H3K4 methyltransferase complexes for transcriptional activation . As showreported . Jpx expKdm5c/Jarid1c and Kdm6a/Utx  and Ddx3x (ATP-dependent RNA helicase), are known as escape genes[KDM5C, KDM6A and DDX3X is associated with cancer development, proliferation, invasion and metastasis in certain type of cancers such as breast cancer [FTX gene has been reported in colorectal cancer, although the downregulation of FTX is also associated with primary breast tumors [Ftx potentially activates Xist in random XCI in mice [Xist knockout in the hematopoietic stem cells leads to hematological cancer in mice [The physiological roles of Xist repeat E, of which mutations affect escape gene expression, remain to be elucidated. Several X-linked genes encoding chromatin modifying enzymes and transcriptional regulators, such as ape genes. Recent ape genes. Aberranape genes. Indeed,t cancer –59. In at tumors ,61. Ftx  in mice . Since p in mice , the strIn conclusion, we provide evidence for the unique role of Xist repeat E for the recruitment of ASH2L and epigenetic and transcriptional regulation of X-linked genes, including escape genes. Further studies on the interaction between ASH2L and Xist RNA though the repeat E will help us to elucidate an underlying mechanism whereby expression levels of escape genes are properly regulated by Xist RNA.Xist expression from tet-inducible Xist cDNA transgene in T20 cell lines was induced by adding 1 μg/ml doxycycline (Dox) in the culture medium for differentiation for 2 days [Xist cDNA transgenes was done as described [16.7 female ES cells were used to generate Xist repeat E deletion mutant . ES cellr 2 days . Cre-Loxescribed .Xist genomic fragment. pBS-sx16delL was transfected into SW106 and targeted by XhoI-SalI fragment of the bacterial targeting vector, generating Xist repeat E targeting vector for mouse ES cells. The Xist repeat E targeting vector linearized by SalI was used for the electroporation using 16.7 female ES cells.We used bacterial homologous recombination system to construct the Xist repeat E targeting vector . Left  as described in Wdr5 coding region as described previously [(F+E) mutation for higher targeting efficiency [Wdr5 locus was confirmed by genomic PCR using primer pair  and DNA sequencing.pSpCas9(BB)-2A-Puro  was used for CRISPR/Cas9-mediated 3xFLAG knock-in to the 5´-end of the eviously ,65. Primficiency . Single-7 cells) were washed with ice-cold PBS and irradiated in 5 ml ice-cold PBS with 400 mJ/cm2 at 254nm. Cells were recovered by scraping with 1 ml RSB buffer  and incubated for 5 min on ice. One quarter of cells (approximately 2x107 cells) were recovered by centrifuge with 1,000 rpm for 5 min at 4°C, snap frozen in liquid nitrogen and store at -80°C until RIP was performed. For formaldehyde-crosslinked RIP, 2x107 cells were incubated in 10 ml PBS/0.5% formaldehyde for 10 min at room temperature. After quenching by final 125 mM glycine for 5 min at room temperature, cells were washed twice by ice-cold PBS, frozen in liquid nitrogen and stored at -80°C until RIP was performed. The UV- or formaldehyde-crosslinked cells were suspended in 0.6 ml SDS lysis buffer . 0.2 ml aliquots each in 1.5 ml tube was sonicated by Bioruptor . After centrifugation with max speed for 10 min at 4°C, the supernatant was pooled in one tube. 0.1 ml aliquot of cell lysate was diluted by 1 ml dilution buffer  and 100 μl was taken as an input. The diluted lysate was incubated with 5 μg of ASH2L antibody  or normal rabbit IgG  at 4°C for 2 hours, and then with 25 μl of Dynabeads protein G (Life Technologies) at 4°C for 1 hour. Magnetic beads were washed twice by ice-cold washing buffer . UV-crosslinked RIP samples and inputs (50 μl) were incubated with 200 μl TE, 0.5% SDS, 0.5 μg/μl Proteinase K, 0.1 unites/ml RNase inhibitor for 1 hour at 37°C. Formaldehyde-crosslinked samples and inputs (50 μl) were reverse crosslinked in rev-crosslinking buffer  for 1 hour at 42°C and then for 1 hour at 55°C. RNA was isolated by Tri Reagent for liquid sample (MRC) with manufacturer’s instruction. RNA was precipitated with 2 μl of GlycoBlue (Life Technologies) and suspended in 20 μl of nuclease-free water. cDNA synthesis and RT-qPCR analysis was performed as described previously except for using different primer pairs 1 , 10  and 11  [Cells at 8 days upon differentiation on 15 cm dish were used for RIP. For UV-crosslinked RIP, cells on 15 cm dish (approximately 8x10GCCCAAG) .Xist, Tsix, Mecp2, Pgk1 on X129 or XCast was performed as described previously [Usp9x and Hprt are as following: Xist repeat E , Usp9x  and Hprt .Allele-specific RT-qPCR analysis using SNP-based allele-specific primers for eviously . In addiHalf-life assay for Xist RNA was performed as described previously .Immuno-FISH was performed as described ,67. Anti129:XCast = 50%:50%). The following primers were used for pyrosequencing experiments is shown in To determine allelic gene expression ratio between 129 and Cast alleles, RT-PCR products amplified from cDNA were analyzed by pyrosequencing analysis by Pyromark Q96 MD (QIAGEN). The relative expression ratio between the two alleles was quantified by the Pyromark MD SNP/Genotyping software according to the manufacturer’s instructions and normalized by the pyrosequencing data obtained from PCR fragment amplified from the genomic DNA  were sequenced by Illumina HiSeq2500. RT-PCR was performed as described previously . Gene-sp7 ES cells (day 0) and differented EB cells (day 12) were crosslinked in 1% formaldehyde/PBS 10 ml at room temp for 10 min and quenched with final 125 mM glycine at room temp for 5 min. After rinse with ice-cold PBS twice and swelling buffer , the fixed cells were resuspended in swelling buffer and incubated on ice for 10 min. The cells were passed through G-25 needle for 10 times, and nuclei were isolated by centrifugation at 1,000 g, for 10 min at 4°C. The nuclei were suspended in sonication buffer  at the concentration of 2x107cells/ml, and divided into 300 μl aliquotes. Sonication were performed in Biorupter sonicator (Diagenode) at power high for 18 cycles, each cycle for 30 seconds on and 30 second off. Cell lysates were centrifuged by table top centrifuge with max speed at 4°C for 10 minutes. The sheared chromatin supernatant were diluted 10 times with dilution buffer . 50 μl of anti-mouse IgG Dynabeads (Life Technologies) were incubated with 5 μg of antibody at 4 oC for 3 hours, and 1 ml diluted chromatin lysate derived from approximately 2x106 cells was rotated with Dynabeads associated with antibody at 4°C overnight. Antibodies used in the ChIP experiments were anti-histone H3K4me3 , anti-K3K27me3  and mouse IgG  as a control. The beads were washed with dilution buffer, wash buffer A , buffer B  and TE, with each buffer twice. The precipated protein/DNA complex were eluted by 200 μl of elution buffer  twince and pooled. The IP and input (100 μl + 300 μl elution buffer) were reverse crossed by adding final 200mM NaCl and incubated at 65°C for 4 hours. The DNA were extracted with phenol/chloroform, precipated by ethanol, and resuspended in 100 μl TE. qRT-PCR analysis was performed as described above with primer pairs in Approximately 1x10S1 Fig(A) We performed RIP experiments using ASH2L antibody  as described in Methods. For western blot, we used anti-ASH2L  and Protein A-HRP . (B) We used the auxin-inducible degron (AID) system to validate the ASH2L antibody. In the presence of the auxin perceptive F-box protein OsTIR1 (E3 ubiquitin ligase), proteins fused with an AID-tag can be induced for rapid degradation by adding auxin. Female ES cells expressing OsTIR1 and minimal AID (mAID)-tagged ASH2L were created by the CRISPR/Cas9-based mAID-tagging method as described ,70, and (AI)Click here for additional data file.S2 FigXist. White and black boxes indicate Xist exons and repeat E, respectively. The targeting vector for Xist repeat E is shown above the map. SA, Splice acceptor; IRES, internal ribosomal entry site; Neo, neomycin resistance gene; tpA, tandem polyadenylation signals. The positions of primer pairs for genomic PCR to confirm the targeting are shown as arrows. (B) Genomic PCR analysis of wildtype (lane 1) and XistΔE  female ES cells to confirm the replacement of Xist repeat E by the Hyg selection cassette. M, NEB 1kb DNA ladder. Black arrow heads indicate the PCR products derived from the XistΔE allele. (C) Strategy to create the XistΔE mutant. SA-IRES-Hyg-tpA cassette flanked by F5 sites were removed by the Flpe expression. (D) Genomic PCR analysis of wildtype (lane 1), XistΔE with the Hyg cassette  and XistΔE without the Hyg cassette  female ES cells to confirm the deletion of Xist repeat E. White arrow head, PCR fragment derived from the wildtype allele. Black arrow head, PCR fragment derived from the XistΔE (+Hyg) allele. Grey arrow head, PCR fragment derived from the XistΔE (-Hyg) allele.(A) Map of the mouse (AI)Click here for additional data file.S3 FigXist/Tsix locus. White and black boxes indicate Xist exons and repeat E, respectively. The targeting vector for Tsix is shown above the map. The positions of primer pairs for genomic PCR to confirm the targeting are shown as arrows. (B) Genomic PCR analysis of wildtype (lane 1), Tsix-truncation (TST) mutant (lane 2) and XistΔE/TST mutant  female ES cells to confirm the Tsix targeting. M, NEB 1kb DNA ladder. Black arrow heads indicate the PCR products derived from the TST mutant allele. (C) Representative phase contrast images of the differentiated embryoid body (EB) cells at day 6 upon differentiation.(A) Map of the mouse (AI)Click here for additional data file.S4 Fig(A) Immunostaining for H3K27me3 (green) and H4K20me1 (red) at day 8 upon differentiation. Nuclei were counterstained by DAPI. Co-localization frequency of H3K27me3 and H4K20me1 at day 8 upon differentiation from three independent experiments. More than 100 nuclei in each ES cell lines at each differentiation time were counted. (B) Colocalization of H3K27me3 (green) and uH2A (red) in differentiating EB cells at day 8 of differentiation. Nuclei were counterstained by DAPI. Scale bar is 10 μm. Colocalization frequency was determined from more than two independent experiments. More than 80 nuclei in each ES cell lines at each differentiation time were counted.(AI)Click here for additional data file.S5 Fig(A) Immuno-FISH for Xist RNA (green) and EZH2 or RING1B (red) at day 0, day 4, and 12 upon differentiation. Nuclei were counterstained by DAPI. Arrowhead indicates colocalized signal of Xist RNA cloud and EZH2 or RING1B. Scale bar is 10 μm. (B) Frequency of EZH2 and RING1B positive cells colocalized with Xist RNA cloud during differentiation. More than 100 nuclei in each ES cell line at each time point were counted in two independent experiments.(AI)Click here for additional data file.S6 FigWdr5 allele. Using primers Wdr5-T7E1-F1 and Wdr5-T7E1-R1, 270 or 336 bp PCR products are amplified from the wild-type or the 3xFLAG knock-in Wdr5 alleles, respectively. (C) Immunostaining for H3K27me3 (green) and ASH2L, RBBP5 or 3xFLAG-WDR5 (red) at day 8 upon differentiation. Nuclei were counterstained by DAPI. Scale bar is 10 μm.(A) Schematics of Wdr5 3xFLAG knock-in by CRISPR/Cas9. The protein coding or 5´-UTR regions are shown as a gray or white box, respectively. The 3xFLAG tag within single-stranded oligodeoxynucleotide (ssODN) is shown as a red box. The first ATG sequence of the translation start is labeled in blue. The protein coding or 5´-UTR regions are capitalized or lowercased, respectively. The sgRNA sequence and adjacent protospacer-adjacent motif (PAM) for CRISPR/Cas9 genome editing are labeled in green and red, respectively. Arrowhead indicates putative cleavage site. (B) Genomic PCR genotyping analysis of the 3xFLAG tag knock-in (AI)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Avian influenza viruses (AIV) potentially transmit to swine as shown by experiments, where further reassortment may contribute to the generation of pandemic strains. Associated risks of AIV inter-species transmission are greater in countries like Nigeria with recurrent epidemics of highly pathogenic AI (HPAI) in poultry and significant pig population. Analysis of 129 tracheal swab specimens collected from apparently healthy pigs at slaughterhouse during presence of HPAI virus H5N1 in poultry in Nigeria for influenza A by RT-qPCR yielded 43 positive samples. Twenty-two could be determined by clade specific RT-qPCR as belonging to the H5N1 clade 2.3.2.1c and confirmed by partial hemagglutinin (HA) sequence analysis. In addition, 500 swine sera were screened for antibodies against influenza A virus nucleoprotein and H5 HA using competition ELISAs and hemagglutination inhibition (HI) tests. Serologically, 222 (44.4%) and 42 (8.4%) sera were positive for influenza A virus NP and H5 antibodies, respectively. Sera reacted to H5N1 and A/H1N1pdm09 strains by HI suggesting exposure of the Nigerian domestic pig population to these viruses. We report for the first time in Nigeria, exposure of domestic pigs to H5N1 virus. This poses potential public health and pandemic risk due to interspecies transmission of avian and human influenza viruses. This is due to the susceptibility of pigs to infection with avian and mammalian influenza viruses, because of possessing both avian  and mammalian  sialic acid receptors for influenza A viruses in their upper respiratory tract. Consequently, pigs could play the role of an intermediate host in the reassortment of genes through inter-species transmission like it happened prior to 2009 influenza pandemic and are therefore important in the emergence of new strains of influenza virus3. Influenza A virus (IAV) genome is composed of eight distinct RNA segments encoding at least 11 proteins which coordinate functions, components and structure of the virus. If two parent viruses co-infect a host cell, they can, through reassortment, exchange genome segments in the progenies. This reassortment of genes and further point mutations are responsible for genetic diversity that gives rise to virus adaptation in new host and emergence of novel and pandemic strains5. One of the greatest public health concerns with enzootic circulation of highly pathogenic avian influenza virus (HPAIV) in agricultural scenarios relates to exposure of humans at the human-animal interface. This bears potential to cause infection in human through direct transmission from infected poultry or through an intermediate host such as pigs6.Scientific investigations predict that avian influenza viruses (AIV) might adapt in swine host and contribute to the generation of a potentially pandemic strain through genetic reassortment between avian and human viruses7.The risks involved in this inter-species transmission of influenza virus and the odd that reassortment of genes in pigs as a mixing vessel would occur is greater in countries like Nigeria with recurrent epidemics of HPAI in regions with high pigs population often in direct contact with poultry. This is because lapses in biosecurity provide ample opportunities for intra- and inter-species co-infections9. Nigeria has also experienced recurrent epidemics of HPAI in poultry, which underscores the likelihood of inter-species transmission of IAV in an epidemiological niche where multiple species co-mingle12. Therefore, the need to monitor IAV ecology and epidemiology predicated a risk-based surveillance carried out in a pig slaughter facility in Nigeria.Shortly after initial cases of pandemic influenza A/H1N1pdm09 reports in human worldwide, the virus was also transmitted to pigs in a reverse zoonotic fashion in many countries including Nigeria13.Apparently healthy domestic pigs from backyard and free range husbandry system in Plateau State, north central Nigeria slaughtered at the Jos central abattoir from 2015–2016 were investigated. Forty three of 129 (33%) tracheal swab specimens collected from these pigs were positive for influenza A virus (M and NP) genes by conventional and RT-qPCR assays. In order to avoid multiple freeze-thawing steps, and to save expensive RT-qPCR material, only samples with lower Cq values were used for further characterisation. Also, not all of these positive samples were investigated in the subsequent tests because of low volume of remaining RNA. Thirty positive samples were tested in multiplex RT-qPCR assays specific for HA and NA subtypes of European porcine influenza viruses. All of them were negative in the HA test , but 26 were positive for N1. Out of the 23 samples tested by the H5N1 duplex assay, 22 were positive in the H5 reaction and two in the N1 part  returned closely related viruses. This included A/Chicken/Nigeria/15VIR399-1/2015, other poultry sequences from Nigeria and from other African countries in 2015 as well as from Asian countries  with 99% sequence homology. As determined with the clade-specific RT-qPCR and by sequence analysis, the swine H5N1 virus genomes detected in this study clustered together and along avian H5N1 clade 2.3.2.1c.14 and new designed and customized primers for N1 based on avian H5N1 sequences from western Africa, 2015. Attempts to isolate the virus from swabs in chicken embryonated eggs and tissues in MDCK (+) cells  were also not successful.Unfortunately, the NA gene sequencing attempts repeatedly failed even with different primer sets including conventional panNA RT-PCR, both variantsTM database and assigned accession number EPI262933 (A/swine/Nigeria/49/H5N1), EPI262932 (A/swine/Nigeria/55/H5N1), EPI262934 (A/swine/Nigeria/73/H5N1), EPI262935 (A/swine/Nigeria/75/H5N1), EPI262950 (A/swine/Nigeria/77/H5N1). Phylogenetically, the topology of the HA tree  were positive by IDvet NP-ELISA. Among the 300 investigated serum samples from 2016, 183 (61%) reacted positive in the IDvet NP-ELISA. Of those, 42 (14%) showed positivity in the IDvet H5 ELISA. All 200 serum samples from 2013 when HPAIV was not present in poultry in Nigeria were negative in the H5-ELISA though 39 (19.5%) were NP positive. HI test against antigen for HPAI H5N1 clade 2.3.2.1.c confirmed H5 antibodies with HI titres ranging from 160 to 1280 in selected 6 of the 42 H5-ELISA positive sera from year 2016. Sera reacted also against LPAI H5N3 virus confirming that the specific component of the sera must be antibodies against H5. No cross reactivity was obtained with H5 HPAI viruses of other clades, i.e. 2.3.4.4b (H5N8) or 2.2 (H5N1). Fourteen NP-positive / H5 negative samples that were tested against swine-specific influenza virus antigens showed positive reaction predominantly for A/H1N1pdm09 with HI titres ranging from 320 to1280. Tables 4.4% were16. In those studies, the virus appeared to have been transmitted from poultry. Our investigation revealed natural exposure of HPAIV H5N1 clade 2.3.2.1.c possibly from infected poultry in Nigeria to domestic pigs as we could find H5N1 RNA in 43 tracheal specimens of swine sampled in December, 2015 to February, 2016 in the slaughter house in Jos, Plateau state, Nigeria. Partial sequencing of the HA showed highest homology to A/Chicken/Nigeria/15VIR399-1/2015 (H5N1) with sequence homology of 99%. HPAIV H5N1 of clade 2.3.2.1c has been circulating in Nigeria since 2015 in poultry and is most prevalent in the Northern region of the country where this study was carried out12. In an RT-qPCR 3plex assay for the detection of swine influenza NA subtypes, it was possible to detect 22 N1-positive samples. However, sequencing of the neuraminidase failed due to the compromised quality of the samples as a result of multiple freeze thawing. Therefore, the sequence identity of the N1 could not be ascertained. Further investigation through surveillance, virus isolation and full genome sequencing is required. Another limitation was the inability to sequence the internal genes due to low quality of the remaining RNAs and low volume of original material which restricted the range of investigations including virus isolation attempts.Infections of swine with AIV in nature seem to be rare, and only very few studies have described detection of HPAIV H5N1 in healthy domestic pigs in Indonesia and ChinaSignificant exposure to influenza viruses was demonstrated serologically with 222 (44.4%) and 42 (8.4%) out of 500 sera samples being positive in the competitive ELISAs for the detection of NP and H5 antibodies, respectively. This was also confirmed by HI assay against H5 and H1 antigens for selected samples. Interestingly, the HI test also indicated cross reactive antibodies in low titres to other serotypes of porcine influenza viruses including H1avN1av, H1huN2, and H3N2 in the study population Table . However17 on co-circulation of avian H9N2 and contemporary human H3N2 viruses, possible inter-species transmission and genetic reassortment in pig was suggested. Also in 2011, influenza A H3N2 variant virus infection in a person who attended an agriculture fair in the USA was detected for the first time and characterised as a reassortant of a SIV H3N2 with A/H1N1pdm0918 which was further spreading among swine and humans in following years20. These reports raised concerns on the possible emergence of zoonotic influenza virus from swine as consequences of inter-species transmission18. Our results provide additional scientific evidence of natural exposure of HPAIV subtype H5N1 currently circulating in Nigerian poultry to pigs in the same epidemiological zone. The risk of possible gene reassortments may be due to presence of different IAV subtypes in domestic animals in the region. Furthermore, this study shows that HPAI H5N1 clade 2.3.2.1c was present in swine population in the study area as also revealed serologically through detection of H5 antibodies.In a study published in 2001et al.21. Mechanical carriage may be possible, but this does not exclude other routes of transmission. Specifically, tracheal swabs (lower respiratory tract) not nasal samples were collected. In addition 8.4% seroconversion to H5N1 in pigs was also observed in the sampled population.We speculate that exposure in swine is due to intensity of AIV outbreaks in the region and the breakdown in control measures. Because AIV detection in swine was not only once and not in the same group of pigs based on different sampling dates and ownership, we can also speculate that exposure was more than once within the epidemiological zone likely due to between-farms transmission similar to observations by Ssematimba et al.22, it was observed that clade 2.3.4.4 HPAIV H5 viruses were poorly adapted for replication and transmission in pigs. In that study, inoculated pigs did not produce HI antibodies. Comparatively, our investigation showed that H5 clade 2.3.2.1c produced high antibody titre in naturally exposed pigs and corroborate observation by Kaplan et al.22, that other lineages of H5 and H1N1pdm09 are more reactive serologically in swine. Though as also observed by Balzli et al.23. AIV may produce lower respiratory tract infection in pigs based on detection of IAV by RT-qPCR in bronchoalveolar lavage fluid (BALF). However poor replication efficiency shown by high Cq values and lack of virus isolation  suggests low adaptation in swine host22. Though domestic pigs showed low susceptibility to HPAI H5N125, genetically engineered reassortants of H5N1 and H1N1pdm09 have been shown experimentally to have potential for mammalian transmissibility in guinea pigs26.In a recent experiment by Kaplan 28. Thus, the large concentration of people and livestock in urban and semi-urban settings provides a veritable ground for emergence and spread of a zoonotic pathogen at the human-animal interface. This was emphasised in a previous study on the prediction and prevention of the next pandemic29 and also verified by serological investigations performed among Nigerian poultry workers which demonstrated H5 reactive sera within this risk group30. In addition, it was also observed that the emergence of zoonotic pathogens that have dominated the pandemics of the past 100 years correlates strongly with human population density and global distribution of animals31.The virus RNA detected in our study were obtained from apparently healthy pigs; asymptomatic and therefore silent transmission of a potentially zoonotic HPAIV in a country with a large population of pigs in intensive and free range husbandry bears public health risks33. If AIV is sustained and adapted in pigs, the consequences comprise suggestions by some investigators35 that a reassortant H5N1 with A/H1N1pdm09 internal genes may have enhanced replication and transmissibility competence compared to the parent H5N1. A previous serosurvey of H5N1 in pigs in Nigeria during 2006–2008 epidemic returned negative results36. However, the viruses that circulated in Nigeria at that time belonged to a different clade of Asian origin H5 HPAIV (clade 2.2.). Sera analysed by HI in the present investigation was negative to that clade further showing that exposure of pigs to H5 virus is a recent event. Thus detection of H5N1 in the current study suggests possibilities of enhanced factors including increased contact rates between avian and swine and widespread H5N1 virus dissemination in the environment, which could aid exposures and biological adaptation in swine host. Furthermore, a large proportion of serum samples showed antibodies against IAV most of which were reactive to A/H1N1pdm09 and therefore calls attention to the theoretical possibility of simultaneous infections with different avian and mammalian influenza A viruses in the population, the basis for reassortment of genes in pigs being a mixing vessel. This also indicates that possibly, human pandemic H1N1 infections (or porcine adapted variants thereof) play the main role for swine influenza in Nigeria. Alternatively, other swine-adapted viruses may circulate which are antigenically distinct from those of Europe or other regions.The significance of our study is the detection of HPAI H5N1 RNA of clade 2.3.2.1c and H5 reactive sera in apparently healthy pigs in Nigeria. Nigeria recorded the highest number of outbreaks of HPAIV in West Africa since 2006 and from 2015 to 2016. Similarly, A/H1N1pdm09 strain is dominant in domestic pigs in the region following 2009 pandemic with seroprevalence in the range of 27.4 to 29% reported in different studiesIn conclusion, the exposure to H5N1 HPAIV from avian to swine, poses yet unknown public health risk if subclinical infection in pigs is sustained and may combine with A/H1N1pdm09 also detected in the population. This might allow the generation of new potentially zoonotic viruses by gene reassortment. The potential of this IAV transmission to humans thus requires further monitoring and investigation of the swine population in Nigeria, especially in regions with high swine density and endemicity of HPAIV in poultry though the risk is up to now theoretical.In the study, 129 tracheal swabs and 129 lung tissues were concurrently and randomly collected through convenience sampling from apparently healthy pigs at a slaughter house in December 2015 through February 2016 in the period of avian influenza epizootic in Nigeria Figs . Differe37. RT real time PCR (RT-qPCR) targeting parts of the nucleoprotein gene were additionally done at FLI, Germany as described by Fereidouni et al.38. Positive samples were thereafter analysed using Ambion-AgPath ID One Step RT-PCR Kit and RT-qPCR protocols for a swine influenza virus (SIV) hemagglutinin (HA) 4plex assay targeting H1pdm, H1avian (H1av), H1human (H1hu) and H3 and for a SIV-neuraminidase (NA) 3plex assay targeting N1, N2, N1pdm14. In addition, an in-house IAV-H5N1 duplex RT-qPCR specific for HAs and NAs of HPAIV H5N1 viruses was carried out . To confirm the high pathogenicity of the viruses in the specimen and to detect the clade of the viruses, RT-qPCR assays a) with a probe specific for HPAI virus HA cleavage sites and b) for the differentiation of 3 H5N1 clades  were also performed13.The swab specimens were initially screened by conventional reverse transcription (RT-) polymerase chain reaction (PCR) for a conserved part of the matrix (M) gene coding region in Nigeria according to the method described elsewhere40. Primers used for HA - GGA ATG ATH GAY GGN TGG TAT GG-HA-(HA-1134-F) and ATA TCG TCT CGT ATT AGT AGA AAC AAG GGT GTT TT-(Bm-NS-890R) targeted a region encoding parts of the HA stalk17. Primers used for NA - (Mix A- ATC GAR GAR TCN TGY – Na-(886.1-F). GCA GTA TAT CGC TTG ACA AGT AGA AAC AAG G-Na (-647-R). Mix B- GTC GAR GAR TGY TCH TGB TA (NA-886.3-F), GCA GTA TAT CGC TTG ACA AGT AGA AAC AAG G (NA-647-R) were described by Gall et al.40. Amplicons were gel purified and sequenced by Sanger sequencing using the BigDye Terminator v1.1 Cycle Sequncing Kit (supplier: Applied Biosystems) at a capillary sequencer  as previously described41. Sequence adjustment were done with Geneious software suite  to generate consensus sequences. Alignments were done using MAFFT (vs7.308)42, alignments were trimmed and realigned (FFT-NS-i x 1000 refinement scoring 200PAM /k = 2). Phylogenetic analysis was done with RAxML43 (GTR GAMMA Model + Rapid bootstrapping 1000 replicates). Query BLAST of the HA sequences in GISAID-EpifluTM (https://platform.gisaid.org/epi3/froned#365177) and NCBI Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM9) was done with sequences from West Africa and sequences of H5N1 obtained from pigs mostly of Asian origin.Amplification was performed for highly conserved parts of HA and NA segments via conventional RT-PCR assays44 against selected reference influenza antigens of European porcine influenza viruses  and against avian influenza viruses [H5N1 clade 2.3.2.1c ; H5N8 clade 2.3.4.4b (A/Tufted duck/Germany/AR8444/16); H5N1 clade 2.2 (A/Whooper swan/Germany/R65/2006); H5N3 , the last one kindly provided by the European Reference Laboratory for Avian Influenza. All laboratory analyses were carried out under standard bio-containment requirement according to level of risks involved and working under biosafety cabinets in BSL-2 and 3 facilities.To assess levels and extent of exposure to influenza viruses in domestic pigs in the study area, 300 recently collected sera (2016) along with 200 archived sera (2013) were analysed for Influenza A, NP and H5 specific antibodies, respectively, using “ID-Screen” Competition ELISA assays obtained from IDVet according to manufacturer’s instructions. A subset of ELISA positive sera were subtyped by hemagglutination inhibition (HI) assayTM - www.platform.gisaid.org/ with the following accession numbers: EPI262932, EPI262933, EPI262934, EPI262935, EPI262950.The datasets generated and/or analysed during the current study are available in the GISAID-EpiFlu


SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three  non-coding and four  coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene.Human  Arabidopsis, worm, fly and mouse [Gene expression is responsible for the synthesis of functional gene products, typically proteins. The question of how many genes are present in the human genome led to the collection of known protein-coding genes not only in the human genome but also in nd mouse –4. The fnd mouse . New prond mouse ,7. A varnd mouse ,9, whichThe serpins (serine proteinase inhibitors) are proteins found in plants, animals, and viruses –12. ThesSERPINA1 gene is located on chromosome 14q31-32.3 [SERPINA1 gene is expressed and translated in different tissues, and encodes alpha1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. The regulation of A1AT expression is controlled by different promoters and transcription start sites located in the 5’UTR of the gene [SERPINA1 gene has 19 transcripts (splice variants), generated in a stimulus- and cell-type specific manner [SERPINA1 transcript variants are produced by the alternative splicing between the non-coding exons 1A, 1B and 1C.Human q31-32.3 , which cthe gene –18. In hthe gene ,19 wherethe gene ,18,20. Oc manner –23. DiffSERPINA1 gene expression found short transcripts lacking exons II or III [SERPINA1 gene exon and intron organization is similar among the species, theoretically short transcripts can also be present in human SERPINA1 gene. In support of our prediction, herein we identified novel short transcripts of SERPINA1 gene and provided evidence for tissue specific expression and regulation of these transcripts.Remarkably, a recent study on ovine I or III . BecauseSERPINA1 transcripts, primers located in exon 1C: 1C_F:5’ctgtctcctcagcttcaggc3’ and 1A: 1A_F:5’tgaggagagcaggaaaggaca3’ were used in combination with a reverse primer located in exon V, EX5_R:5’ccatgaagaggggagacttgg3’. The PCR was performed under the following conditions: 35 cycles of 94°C for 45s, 60°C for 45s, and 72°C for 45s. Amplified products were visualized in 1–2% agarose gels. PCR products were purified by using PCR purification Kit (Qiagen) and subsequently cloned into pGEM-T easy vector (Promega). Ligation reactions were used to transform DH5α competent cells. Clones containing PCR products were selected by blue/white colony screening and standard ampicillin selection. Positive transformants were analyzed by the PCR and sequenced using SERPINA1 primers.Peripheral blood samples were obtained from subjects previously tested for A1ATD, and informed consent for this study was signed. The study was approved by the ethics committee of Instituto de Salud Carlos III. RNA isolation from peripheral blood leukocytes was performed using RNAeasy kit (Quiagen) following manufacturer’s recommendations. The cDNA synthesis was generated using the Maxima First Strand cDNA Synthesis kit (Thermo Scientific). To amplify 5’tgggacagtgaatc/gtctgc3’ or the spanning region between 1C-exon5, ST1C5v3_F:5’acctgggacagtgaatc/gccgtg3’ in combination with a reverse primer in exon 5 for both reactions EX5_R:5’ccatgaagaggggagacttgg3’. Specific fluorescent-labelled Taqman probes were selected from the Universal probe library, UPL, Roche. UPL probe #57 was used for the ST1C4 transcript and the probe #3 was selected for the ST1C5 transcript. GUSB gene was used as an endogenous control to normalize the expression levels of the transcripts. All experiments were performed in triplicate and data were analyzed by Applied Biosystems 7500 software v2.0. Relative expression was calculated by using the comparative Ct method and obtaining the fold-change value (ΔΔCt).The expression levels of two short transcripts, ST1C4 and ST1C5, were independently analyzed by real time quantitative PCR (qPCR). cDNA samples were diluted and amplified using Taqman Fast advance master mix in a 7500 Fast Real Time PCR System (Applied Biosystems) with recommended PCR cycle conditions. To amplify the ST1C4 or ST1C5 transcripts two different reactions were performed using boundary-spanning primers for the sequence covering the 1C-exon4 junction ST1C4_F:SERPINA1 full transcripts and compared these with expression of identified short transcripts. We individually analyzed alternative full transcripts generated by the use of exons 1A, 1B and 1C by using primers and methods reported before [We also analyzed d before .We used a set of first-strand cDNAs from different tissues for analysis of short transcripts ST1C4 and ST1C5. We analyzed 16 commercially available first-strand cDNAs from 16 different human tissues  as described before . We perf6 cells/ml in RPMI-1640 with 2 mM N-acetyl-L-alanyl-L-glutamine  supplemented with 1% nonessential amino acids, 2% sodium pyruvate and 20 mM HEPES and plated. We incubated cells for 75 min at 37°C and 5% CO2 to allow monocytes to adhere to the cell culture plates. Afterwards, we removed non-adherent cells by washing with PBS containing Mg2+ and Ca2+  and fresh medium was added. Next day, adherent PBMCs were used for experiments.Human Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy volunteers using Lymphosep discontinuous gradient centrifugation. PBMCs were re-suspended at a density of 5 x 106 per well) were always left alone for 30–40 min in cell culture plates pre-coated with fetal calf serum.Neutrophils were isolated from the freshly obtained peripheral blood of healthy donors using PolymorphoprepTM  according to the manufacturers’ recommendations. The neutrophil purity was typically 98% as determined by cytospins and cell viability was more than 90% according to trypan blue staining. Before starting the experiment, neutrophils  with 10 kDa molecular weight cut-off (MWCO), to remove any large protein and concentrate the C-terminal peptides. 100 μL aliquots of both standard solutions and real samples were acidified by addition of 2 μL of formic acid (FA), to reach a final pH of about 3.0 was reached, and analyzed by LC-MS.C-10, C-21 and C-36 standard peptides  were dissolved in RPMI Medium, to obtain a final concentration of 20 μM. All supernatants from neutrophil cultures (controls and LPS-stimulated) were filtered using Microcon18 column  using a linear gradient (2–60% solvent B in 60 min) in which solvent A consisted of 0.1% aqueous FA and solvent B of acetonitrile (ACN) containing 0.1% FA. Flow-rate was 0.2 mL/min. Mass spectra were generated in positive ion mode under constant instrumental conditions: source voltage 5.0 kV, capillary voltage 46 V, sheath gas flow 40 (arbitrary units), auxiliary gas flow 10 (arbitrary units), sweep gas flow 1 (arbitrary units), capillary temperature 200°C, tube lens voltage –105 V. MS/MS spectra, obtained by CID studies in the linear ion trap, were performed with an isolation width of 3 Th m/z, the activation amplitude was 35% of ejection RF amplitude that corresponds to 1.58 V. All analyses were performed in triplicate.The LC-MS system  consisted of a thermostated column oven Surveyor autosampler controlled at 25°C; a quaternary gradient Surveyor MS pump; a diode array detector and a Linear Trap Quadrupole (LTQ) mass spectrometer with electrospray ionization ion source controlled by Xcalibur software 2.0.7. Analytes were separated by RP-HPLC on a Jupiter  CStatistical Package  was used for the calculations. An independent two-sample t-test was used. The differences in the means of cell experimental results were analyzed for their statistical significance using one-way ANOVA combined with a multiple-comparison procedure (Scheffe multiple range test), with an overall significance level of p = 0.05.SERPINA1 expression we prepared cDNA from human peripheral blood leukocytes and used a primer pair for a full-length SERPINA1 cDNA (1C_F and EX5_R) to amplify SERPINA1 transcripts. When agarose gels visualized amplified products, we identified an expected 1279 bp band corresponding to the full-length A1AT. Cloning and sequence analyses confirmed the presence of the exon 1C and four coding exons (exon II to exon V) encoding the 419 aa A1AT protein. This transcript corresponded to the Ensembl transcript ENST00000393087.Peripheral blood leukocytes  are readily accessible source of information about the health and physiological state of an individual. By comparing gene expression profiles of leukocytes, it is possible to identify genes with cell-type-specific expression patterns . To analSurprisingly, in addition to the 1279 bp band, we constantly observed two bands of few hundred base pairs, one of which (a band of 358 bp) revealed a short transcript of A1AT . After cSince SERPINA1 alternative splicing events typically occur among the first non-coding exons, we investigated whether the newly identified short transcripts can possibly originate from exon IA. Using a primer pair located in exon IA (1A_F) and exon V (EX5_R), three main bands and few less evident bands occurred, similarly as we found by using the primer at exon IC (1C_F) . The banSeveral colonies expressing an intermediate band of 367 bp were obtained, and sequencing analysis revealed a transcript structure containing exon IA directly bound to exons IV and V (ST1A4) . FinallySERPINA1 gene expression also found short transcripts lacking exons II or III [SERPINA1 gene identified in this study. Importantly, however, we found a RNA-Seq data set [A previous study on ovine I or III . Howeverdata set , presentInterestingly, in silico analysis showed that, ST1C4 and ST1C5 as well as ST1A4 and ST1A5 have an ORF in exon V starting at Met351 of the full-length A1AT protein. Further analysis confirmed that in the exon V, in addition to Met351, there are three more methionine residues. Hence, putative peptides generated by the short transcripts would have the same C-terminal amino acid sequence as a full-length A1AT protein, i.e. the last 44aa, 36aa, 21aa or 10aa, respectively .SERPINA1 gene have a tissue specific expression, we performed a quantitative analysis focusing on ST1C4 and ST1C5, using cDNA from various human tissues  as part of the reactive site loop of the A1AT, which acts as a docking site for binding and neutralizing neutrophil elastase [So far, all described alternative human elastase . Therefoelastase , was fou site Met8-Ser359 http://merops.sanger.ac.uk/index.shtml). In both cases, interaction between A1AT and proteases results in cleavage of A1AT and irreversible loss of its inhibitory activity. Different studies provide evidence that native A1AT is proteolytically processed into firmly attached larger N-terminal and shorter C-terminal fragments [de novo synthesized and released by different cells.A1AT is a well-known protease inhibitor forming stable complexes with a range of serine proteases, specifically neutrophil elastase and proteinase 3. A1AT can be targeted by non-specific proteases  without the formation of stable inhibitor complexes. (MEROPS database, ragments . Interesragments ,37–41. Tragments , extraceragments , pro- orragments , and inhragments . We prevragments . We alsoragments . Hence, In summary, we identified novel short transcripts of the SERPINA1 gene, differentially expressed in tissues and cells. We also provide experimental evidence that the expression of short transcripts can be regulated and that C-terminal peptides of A1AT can be found in free form in LPS stimulated neutrophils.


The expression level and clinical significance of NR4A1 are presently unknown in the non-small-cell lung carcinoma (NSCLC). This study aimed to explore the expression, prognostic value, and function of NR4A1 in NSCLC. Methods: Clinicopathological parameters of 167 NSCLC patients who received radical surgery from January 2007 and December 2012 were retrospectively reviewed. The NR4A1 expression in NSCLC tumors and the adjacent matched para-carcinoma specimens were examined, and the association between NR4A1 expression and clinical variables was explored. Cell viability assay, and transwell migration and invasion assays were used to access the function of NR4A1 in NSCLC. Kaplan-Meier analysis and Cox regression were performed to investigate the prognostic significance of NR4A1 for NSCLC. Results: NR4A1 was overexpressed in NSCLC tissues compared with the para-carcinoma specimens. Consistently, Oncomine analysis showed that NR4A1 was overexpressed in NSCLC tissues compared with normal tissues in published datasets (P < 0.001). The elevated NR4A1 expression was associated with carcinoma recurrence (P < 0.05). The 5-year median overall survival (OS) and progression free survival (PFS) were significantly poorer in the NR4A1-overexpression group. Multivariate Cox analysis showed that NR4A1 overexpression was an independent factor for OS  and PFS  in NSCLC. Moreover, knockdown of NR4A1 significantly reduced NSCLC cell proliferation, migration, and invasion. Conclusions: NR4A1 exhibits a tumor-promoting effect on NSCLC, and might serve as a promising prognostic biomarker and a therapeutic target for NSCLC. Lung carcinoma is one of the leading causes of malignancy-related death worldwide due to its high frequency and metastatic potential . Non-smaThe orphan nuclear receptor 4A1  is a member of NR4A orphan receptors , which pIn the current study, a retrospective study was conducted to evaluate the expression and prognostic value of NR4A1 in NSCLC, and role of NR4A1 in NSCLC was also explored. Our data suggested that NR4A1 might be a useful predictor and therapeutic target for NSCLC.The clinicopathological parameters of NSCLC patients were summarized in Table Results of qRT-PCR analysis showed that the NR4A1 mRNA was overexpressed in NSCLC carcinoma tissues compared with the matched para-carcinoma tissues Figure . ConsistThe clinicopathological parameters were further compared based on the NR4A1 protein expression level. As showed in Table in vitro study. NR4A1 expressions were remarkably suppressed in shRNA-NR4A1 group of SPC-A1 and H1299 cells . Western blotting and real-time PCR results showed that higher NR4A1 protein and mRNA expression were detected in SPC-A1 and H1299 Figure , which ws Figure . CCK-8 as Figure . These dCompared with the scramble-shRNA group, the shRNA-NR4A1 group showed remarkably decreased migration and invasion ability , advanced clinical stage (P = 0.001) and carcinoma recurrence (P = 0.033). The NR4A1 protein expression was further identified as independent prognostic factors for PFS and OS .et al. showed that downregulation of NR4A1 decreased colon cancer cell growth, induced apoptosis, decreased expression of survivin and other Sp-regulated genes, and inhibited mammalian target of rapamycin signaling [et al. reported that NR4A1 could promote breast cancer invasion and metastasis via activating TGF-β signaling [Nuclear receptors including NR4A receptors are of integral importance in carcinogenesis . Althougignaling . Zhou etignaling . Interesignaling . Wu et aignaling . These rThe potential limitations including: the relative small size population limited the degree of evidence. Accordingly, larger and muti-center clinical study needs to be conducted to validate our finding. Besides, our results showed that NR4A1 exhibits oncogenic activity in NSCLC, but the underlying molecular mechanisms still need to be elucidated in further studies.NSCLC patients receiving radical surgery at the Second Affiliated Hospital of Chongqing Medical University, who were confirmed histologically from January 2007 and December 2012, were enrolled in this study. Exclusion criteria: 1) patients with distant metastasis found pre-operation or during the operation; 2) patients who received treatment pre-operation. Totally, 167 patients with complete clinical data and follow up information were enrolled in this study. The clinical information was collected, including patient age, gender, tumor size, clinical stage, histological type and survival data. Outpatient visit or telephone-based follow-up was conducted. The median follow-up time was 63.1 months. Overall survival (OS) was defined as time from the day of diagnosis to the day of last visit or death. Progression free survival (PFS) was defined as the time elapsed between treatment initiation and tumor progression or death from any cause. Written informed consents were obtained from all participants according to the Helsinki Declaration, and this study protocol was approved by the Ethics Committee of our hospital (No. SAHCMU1207).NSCLC cell lines  and HEK293T cells were purchased from the Cell Bank of Shanghai Biology Institute, Chinese Academy of Science) and cultured in RPMI-1640  at 37°C in a humidified incubator under 5% CO2 conditions. 10% fetal bovine serum  and 1% penicillin/streptomycin (Life Technologies) were supplemented into the culture media. shRNA-NR4A1 (5′-CGCGTAAATCCACCCCGCTCAGCTGTTTGGAAAGAGATTTGCTCGGTTCCAAAAGC-3′) and scramble-shRNA were synthesized  and transfected into pLVTHM vector . Then, the lentiviral packaging plasmid was transfected into HEK293T cells with Lipofectamine 2000  according to the manufacturer’s protocol. Lentiviruses were collected at 48 or 72 hours after transfection.4 cells/well. Cell viability was detected using Cell Counting Kit-8  following the manufacturer’s instructions. The optical density values at 450 nm were measured using a microplate reader .Cells were seeded in 96-well plates at a density of 1 × 10Cell migration and invasion assays were performed using Boyden chambers  containing polycarbonate filters. Cells transfected with the indicated virus were serum starved for 24 h and seeded in the upper chamber; then, medium containing 20% FBS was added in the lower chamber. After 24 h of incubation, cells on the upper side of the filter were completely removed. The remaining cells were fixed inparaformaldehyde and stained with 0.05% crystal violet staining solution. The cells on the lower surface of the filters were defined as migrating cells and counted under the microscope . Invasion assays were also performed in Boyden chambers with polycarbonate filters coated with Matrigel  on the upper side. The rest was performed as migration assay.-ΔCt method.RNA was isolated and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed using SYBR Green polymerase chain reaction master mix according to the manufacturer’s instructions . Primers for qRT-PCR were as follows: NR4A1, forward 5’- CAGCTTGCTTGTCGATGTC-3’, reverse 5’- GTGTCCATGAAGATCTTGTCAATG-3’; D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5’-CGAGCCACATCGCTCAGACA-3’, reverse: 5’-GTGGTGAAGACGCCAGTGGA-3’. The reactions was incubated at 94°C for 30 seconds, followed by 38 cycles of denaturing at 95°C for 5 seconds, annealing at 60°Cfor 45 seconds. The relative amount of NR4A1 mRNA was normalized to GAPDH. The results of qRT-PCR were analyzed by using the 2Frozen tissue was homogenized in lysis buffer containing protease inhibitors. The supernatant was collected after centrifugation . The cell lysates or tissue homogenates were centrifuged for 10 min . The supernatant was collected, and the protein concentration was calculated with a Pierce BCA protein assay kit . Equivalent quantities of proteins were separated on 10% SDS–PAGE gels and transferred to PVDF membranes . After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were immunostained with primary antibodies at 4°C overnight, and washed three times in TBST followed by incubation with the secondary antibody for 3 h. Band signals were detected with an enhanced chemiluminescence reagent . The following primary antibodies were used: NR4A1  and GAPDH . The protein levels were normalized to those of GAPDH. The NR4A1 protein levels were grouped into high NR4A1 protein expression group and low NR4A1 protein expression group based on the median expression level.Human tissue sections were deparaffinized with xylene and rehydrated through addition of ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced antigen retrieval was carried out for all sections in 0.01 M sodium citrate buffer, pH 6 at 94°C for 30 min. Human NR4A1 antibody  was applied and incubated at 4°C overnight. The antibody-binding signal was detected using the NovoLink Polymer Detection System (Leica Microsystems) and visualized with the diaminobenzidine reaction. The sections were counterstained with hematoxylin. The immunohistochemistry results were scored by taking into account the percentage of positive detection and intensity of the staining.https://www.oncomine.org). P values < 0.05 were considered statistically significant.Data analysis was performed with the software package SPSS version 20.0 . All experiments were independently repeated at least three times. All data are expressed as the mean ± standard deviation (SD). The categorical variables were analyzed by a chi-squared test. Kaplan-Meier method and the Multivariate analyses were conducted to identify significant independent factors for the prognosis. NR4A1 expression in human NSCLC in published datasets was analyzed using Oncomine (In summary, we showed that NR4A1 expression is elevated in NSCLC, and high expression of NR4A1 is associated with poor survival of NSCLC patients. Moreover, downregulation of NR4A1 represses NSCLC cell proliferation, migration, and invasion. Our data suggest that NR4A1 might be an appealing prognostic biomarker and a therapeutic target for NSCLC.


Similarly, co-expression of Cx40 Q236H with Cx43 resulted in a significantly lower Gj. Transjunctional voltage-dependent gating (Vj gating) properties were also altered in the GJs formed by Q236H. Reduced GJ function and altered Vj gating may play a role in promoting the Q236H carriers to AF.Atrial fibrillation (AF) is the most common form of cardiac arrhythmia. Recently, four novel heterozygous Cx40 mutations—K107R, L223M, Q236H, and I257L—were identified in 4 of 310 unrelated AF patients and a followup genetic analysis of the mutant carriers’ families showed that the mutants were present in all the affected members. To study possible alterations associated with these Cx40 mutants, including their cellular localization and gap junction (GJ) function, we expressed GFP-tagged and untagged mutants in connexin-deficient model cells. All four Cx40 mutants showed clustered localization at cell–cell junctions similar to that observed of wildtype Cx40. However, cell pairs expressing Cx40 Q236H, but not the other individual mutants, displayed a significantly lower GJ coupling conductance (G Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia affecting millions of people worldwide ,2. With GJ channels are composed of connexins. In humans, there are 21 different connexins and all of them share a similar topological structure of four transmembrane domains (M1–M4), two extracellular domains (E1 and E2), one cytoplasmic loop (CL), and both the amino terminus (NT) and carboxyl terminus (CT) lie within the cytoplasm ,16. Six GJA5) have been linked to AF patients and families [GJA5 in four of 310 unrelated AF patients, resulting in heterozygous missense mutants in Cx40 protein: K107R, L223M, Q236H, and I257L [The importance of Cx40 and Cx43 in the heart has been highlighted in animal models and genetic mutation studies. Mice with an ablation of Cx43 in the heart develop ventricular arrhythmias leading to sudden cardiac death . An in vfamilies ,12,13,14nd I257L . Furthernd I257L . FunctioExpression of AF-linked Cx40 mutants  was used to study their localization in live HeLa cells. As shown in js) of cell pairs expressing each of the Cx40 mutants and wildtype Cx40 are presented (p > 0.05 for each of the mutants). The coupling conductance (Gj) of cell pairs expressing K107R, L223M, or I257L was also not different from that of wildtype Cx40 (j was observed in cell pairs expressing Q236H (p < 0.05).Dual whole-cell patch clamp was used to study the functionality of untagged AF-linked Cx40 mutants in N2A cell pairs. Representative junctional currents (Iresented A. The avype Cx40 C. Howevej gating) of AF-linked Cx40 mutants, we measured Ijs in cell pairs in response to a series of Vj pulses  when Vjs ≥ 40 mV  of each mutant (filled circles) or wildtype Cx40 (open grey circles) was plotted at different Vjs  at each of the tested Vjs , except Q236H GJ that showed consistently lower τs at all tested Vjs (p < 0.05).To analyze Vsted Vjs A. The avjs that were not statistically different from those of wildtype Cx40:Cx43 (j of cell pairs co-expressing Q236H:Cx43 was significantly lower than that of wildtype Cx40:Cx43 (p < 0.05).To investigate if AF-linked Cx40 mutants had a trans-dominant negative effect on wildtype Cx43, each of the mutants was co-expressed with Cx43 (with an untagged DsRed). Cell pairs successfully expressing both connexins were selected for dual whole-cell patch clamp. Cell pairs successfully co-expressing K107R:Cx43, L223M:Cx43, or I257L:Cx43 showed coupling percentages and Gx40:Cx43 B,C. The js than wildtype Cx40 when expressed alone or co-expressed with Cx43. To further test whether Q236H could affect the function of heterotypic Q236H/Cx40 GJs, we mixed cells expressing Q236H (with untagged GFP) with cells expressing Cx40 (with untagged DsRed) and performed dual patch clamp on heterotypic cell pairs  (Propidium iodide (PI) uptake assay was used to investigate the hemichannel function of AF-linked Cx40 mutants as elevated PI uptake was observed in AF-linked Cx40 mutants, including L221I ,39. Figu< 0.001) B. These j) when expressed alone or together with Cx43. The GJs formed by each of these mutants also failed to show any obvious change in the Vj gating properties. In contrast, Q236H GJs exhibited a significantly reduced Gj when expressed alone or together with Cx43. In addition, Q236H GJs also showed altered Vj gating, specifically a reduction in the Vj required to close the channel (V0) and an increase in Vj-gating kinetics. These defects associated with Q236H might play a role in the pathogenesis of AF in the mutant carriers.In this study, we examined morphological and functional characteristics of four recently identified AF-linked Cx40 mutations  in vitro. Our localization experiments showed that YFP-tagged K107R, L223M, Q236H, and I257L were able to form GJ plaque-like structures at the cell–cell interface in HeLa cells, similar to that of wildtype Cx40. PI uptake by each of these mutant (untagged)-expressing cells showed no significant increase from that of Cx40, indicating no increase in hemichannel activity in any of the mutant-expressing cells. Dual patch clamp experiments revealed that the Cx40 mutants  showed no apparent change in coupling conductance (GGJA5 gene (encoding Cx40) have been identified in AF patients with genetic predisposition [j) was observed in G38D, I75F, A96S, and Q236H GJs, probably due to impairment at the GJ channel [j levels of G38D and A96S GJs have been reported [j of heterotypic mutant/wildtype GJs, altered Vj gating properties of homotypic  or heterotypic  Vj gating properties, a substantially reduced open probability without changing unitary channel conductance (I75F), or elevated unitary channel conductance (G38D and M163V) [So far, a total of ten germlines and three somatic mutations in the coding region of the position ,14,37,40position . Similarposition . Functioposition ,41. (2) position ,14,38,39 channel ,42. Some channel ,42. Althreported , the majreported . (3) Detd M163V) . (4) Onld M163V) . (5) Thed M163V) ,45,46,47j) of AF-linked Cx40 mutants due to either an impaired localization or GJ channel function is known to reduce the action potential conduction velocity [j reduction not only in the mutant GJs but also when they are co-expressed with wildtype Cx40 and/or Cx43 [j when expressed alone and together with Cx43. We did not perform co-expression of this mutant with wildtype Cx40 because our untagged Cx40 construct (Cx40-IRES-DsRed) had a very low transfection efficiency. Second, enhanced Vj gating by lower Vjs and faster gating kinetics by Cx40 mutants could also dynamically down-regulate Gj when sufficient junctional delays exist [j gating with a minimum conductance level reaching a quarter of the maximum Gj [0, and faster Vj-dependent deactivation kinetics of Q236H mutant GJs predict an increased Vj gating when sufficient junctional delay exists. It is not clear whether Vj gating of Cx40 or Q236H GJs could dynamically down-regulate Gj as observed for Cx45 GJs [j) in different possible atrial GJs. At present, we cannot rule out other changes, such as biosynthesis, and turnover of the mutant Cx40 protein may also change the abundance and function of Cx40 at the intercalated discs, which may need genetically modified animal models to assess fully.As discussed above, there is a variety of molecular/cellular changes associated with AF-linked Cx40 mutants. Whether these molecular/cellular changes play a role in the pathogenesis of AF is not clear. Several theoretical possibilities exist. First, a reduced macroscopic coupling conductance . It is possible that one or more of these mutants are benign mutants that do not necessarily play a role in promoting AF, especially at the least conserved position, I257. (2) Both of our model cells, HeLa and N2A cells, are convenient model systems to study localization, function of GJs, and hemichannels. They are GJ deficient, easily transfected with connexin mutant constructs, and easily accessible for morphological, dual patch clamp, or dye-uptake experiments. However, these cells are not cardiomyocytes and may not recapitulate all aspects of GJs at the intercalated discs of cardiomyocytes and, therefore, some defects might go undetected. Future studies on cell models that are closer to atrial myocytes will likely help to resolve the role of these Cx40 mutants linking to AF. (3) We have good rationales to focus our study on the morphology and functional changes in GJs and hemichannel activities. However, there are other unconventional functions of connexins, including but not limited to aggregation of protein complexes at the cell–cell junctions, adhesion, cell growth control, and differentiation, etc., that require specific biological assays to evaluate.In our present study, we did not detect any obvious defects in GJ distribution, function, or hemichannel activities in three out of four AF-linked Cx40 mutants . It is not clear how these mutants relate to AF. Here are some possibilities. (1) These mutants are located in the CL (K107R), M4 (L223M), and CT (I257L) domains of Cx40. The CL and CT domains of Cx40 show a lot more residue variation in different vertebrate species than the E1, E2, and M1–4 domains. Following this general trend, the conservation percentage at these residue positions in Cx40 are L223 (in M4 domain) 85%, K107 (in CL domain) 74%, and I257 (in CT domain) 28% across 47 different species  were created as previously described ,59. The K107RForward: 5′ CAGGAGAAGCGCAGGCTACGGGAGGCC 3′Reverse: 5′ GGCCTCCCGTAGCCTGCGCTTCTCCTG 3′L223MForward: 5′ CTCCTCCTTAGCATGGCTGAACTCT 3′Reverse: 5′ AGAGTTCAGCCATGCTAAGGAGG 3′I257LForward: 5′ CCCTCTGTGGGCCTAGTCCAGAGCTGC3′Reverse: 5′ GCAGCTCTGGACTAGGCCCACAGAGGG3′Q236HForward: 5′ GGAAGAAGATCAGACACCGATTTGTCAAACC3′Reverse: 5′ GGTTTGACAAATCGGTGTCTGATCTTCTTCC3′All these Cx40 mutant constructs were sequenced to confirm the accuracy of the nucleotide sequence.2. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum, 1% penicillin, 1% streptomycin, 4.5 g/L d-(+)-glucose, 584 mg/L l-glutamine, and 110 mg/L sodium pyruvate. Twenty-four hours before cell transfection, N2A or HeLa cells were replated into a 35-mm dish at 60% confluency. Transfection was performed the next day by adding 0.8–1 μg of DNA with 2 μL of the transfection reagent X-tremeGENE HP DNA . To assess the effect of Cx40 mutants on wildtype Cx43, N2A cells were transfected in a 1:1 ratio of Cx40 mutants-IRES-GFP and Cx43-IRES-DsRed. Cell pairs successfully co-expressing both GFP and DsRed were selected for measuring coupling conductance with dual whole-cell patch clamp (see below).Connexin-deficient mouse neuroblastoma (N2A) and human cervical carcinoma (HeLa) cells  were cultured at 37 °C with 5% COHeLa cells were transiently transfected with YFP-tagged Cx40 mutants. One day after transfection, cells were replated on 10 mm glass coverslips and incubated overnight. The number of successfully transfected cell pairs forming GJ plaque-like structures at the cell–cell interface were counted. A confocal microscope (Zeiss LSM800 with Airyscan)  was used to observe mutant-YFP and wildtype Cx40-YFP localizations as described earlier .2, 2 mM CaCl2, 1 mM BaCl2, 2 mM CsCl, 2 mM Na Pyruvate, and 5 mM d-glucose with pH and osmolarity of 7.4 and 310–320 mOsm, respectively. The recording chamber was placed on an upright fluorescent microscope  to visualize reporter (GFP)-positive cell pairs. Patch pipette was filled with intracellular solution (ICS) containing 130 mM CsCl, 10 mM EGTA, 0.5 mM CaCl2, 4 mM Na2ATP, and 10 mM Hepes with pH 7.2 and osmolarity of 290–300 mOsm. Dual whole-cell patch clamp technique was performed on isolated cell pairs expressing the Cx40 mutant. Initially, cell pairs were both voltage clamped at 0 mV. Then, a 20 mV voltage pulse was applied to one of the cell pairs (pulsing cell) while keeping the other clamped at 0 mV (the recording cell). If functional GJ channels exist between the cell pairs then a transjunctional current (Ij) can be measured at the recording cell via MultiClamp 700A  and stored in a PC via an AD/DA interface (Digidata 1322A) and pClamp9.2 software . Gj was calculated (Gj = Ij/Vj). Vj gating properties were studied by applying a series of voltage pulses (±20 to ±100 mV in 20 mV increment) as described in our previous studies [On the experimental day, transfected N2A cells were replated onto glass coverslips and incubated for 1.5 to 3 h. Coverslips with cells were transferred into a recording chamber and bathed in extracellular solution (ECS) containing 135 mM NaCl, 5 mM KCl, 10 mM Hepes, 1 mM MgCl studies ,56.2, 2 mM CaCl2, 10 mM HEPES, and 25 mM d-(+)-glucose adjusted to pH 7.35 and osmolarity of ~298 mOsm. HeLa cells were incubated in a divalent cation-free extracellular solution (DCF-ECS) containing PI (150 μM). Removal of both Ca2+ and Mg2+ ions, as well as including EGTA (2 mM) in the DCF-ECS, were used to facilitate PI uptake via GJ hemichannels. After incubation at 37 °C for 15 min, cells were washed three times with DCC-ECS at room temperature prior to observation with a fluorescent microscope . The number of transfected HeLa cells with or without PI uptake was counted and the percentage of cells with PI uptake was calculated. Only isolated individual HeLa cells were counted to prevent errors caused by GJ channels in cell clusters.AF-linked Cx40 mutants hemichannel function were assessed using propidium iodide (PI) uptake assay. HeLa cells were transiently transfected with each of the Cx40 mutants in an IRES-GFP vector . We usedj) using dual patch clamp, and the percentage of cells displaying PI uptake for the hemichannel study. One-way ANOVA followed by Tukey post-hoc test was used to compare the Boltzmann fitting parameters of each mutant and wildtype Cx40 at the corresponding Vj polarity. Statistical significance is denoted with different levels of significance .Mann–Whitney U test was used to compare each of the mutants against wildtype Cx40 for the percentage of cell pairs with morphological GJ plaques, the coupling percentage and conductance (Gj when expressed alone or with the wildtype Cx43, and altered Vj-gating kinetics and the V0, which might play a role in AF pathogenesis. The other mutants—K107R, L223M, and I257L—did not exhibit any apparent GJ or hemichannel functional impairments in our cell models.In summary, our results indicate that the AF-linked Cx40 Q236H mutation exhibited GJ function impairment by reducing the overall G


The MSA1 protein is localized to the nucleus and is required for both S-adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over-accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation.Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the  more sulphur accumulation1 (msa1-1) and demonstrate the function of MSA1 in controlling sulphur accumulation in Arabidopsis thaliana. The MSA1 protein is localized to the nucleus and is required for the biosynthesis of S-adenosylmethionine (SAM) which is a universal methyl donor for many methylation reactions, including DNA methylation. Loss of function of MSA1 reduces the SAM level in roots and affects genome-wide DNA methylation, including the methylation of sulphate transporter genes. We show that the high sulphur phenotype of msa1-1 requires elevated expression of the sulphate transporter genes which are differentially methylated in msa1-1. Our results suggest a connection between sulphur homeostasis and DNA methylation that is mediated by MSA1.Sulphur is an essential element for all living organisms including plants. Plants take up sulphur from the soil mainly in the form of inorganic sulphate. The uptake of sulphate and assimilation of sulphur have been well studied. However, the regulation of sulphur accumulation in plants remains largely unknown. In this study, we characterize the high leaf sulphur mutant  O-acetylserine (thiol) lyase (OAS-TL) catalyzes the condensation of sulphide and O-acetylserine (OAS) to form cysteine (Cys), the first organic-reduced sulphur compound. Cys then serves as a precursor for the biosynthesis of methionine (Met), glutathione (GSH), vitamins and other sulphur derivatives. Met can be further used for the biosynthesis of S-adenosylmethionine (SAM) which is a universal methyl group donor for many methylation reactions -L-serine and monoglutamylated tetrahydrofolate as substrates as previously described in detail [To clone tor pHUE  as histitor pHUE . Adequattor pHUE  and usedtor pHUE  using 3-n detail .MSA1 for SHM activity assay using hexaglutamylated THF as the substrate, total RNA was isolated from the leaf of Col-0 using the RNeasy Plant Mini Kit , and the cDNA was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) and an oligo78 (dT) primer. The open reading frames were then amplified using Taq2000 polymerase  using the primers listed in E. coli (Novagen) to express the recombinant proteins. Bacteria were cultured at 37°C in LB medium containing 100 μg ml-1 kanamycin and 34 μg ml-1 chloramphenicol. When an optical density at 600nm of 0.6–1 was reached, isopropyl-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the incubation continued overnight at 15°C. The induced bacteria were pelleted at 5,000 g for 15 min at 4°C. The collected bacteria were frozen in liquid N2 and stored at −80°C until use. The recombinant enzymes were purified from the bacterial lysate using an Äkta FPLC system equipped with 1-ml IMAC column  charged with Ni2+ according to the manufacturer’s protocol. All chromatography steps were performed at 4°C. A charged column was equilibrated with Binding Buffer  before loading the clarified cell lysates. Unbound proteins were removed by washing with 15 column volumes of Binding Buffer, followed by elution of bound proteins by a linear gradient of Binding Buffer to Elution Buffer  over 15 column volumes. Both recombinant proteins eluted at about 200 mM imidazole. Fractions containing the recombinant proteins were pooled and immediately desalted into Protein Storage Buffer  using PD-10 desalting columns . The desalted samples were aliquoted and stored at -80°C until use. The enzymes were assayed at 30°C for 20 min with 10 μM hexaglutamylated THF and 5 mM serine as substrates using an HPLC-based fluorometric assay as described before [-1 mg-1 protein. No product peaks were visible in any of the blanks or assays.To clone d before . The resE. coli glycine auxotroph (GS245(DE3)pLysS; shmt-) was performed as previously described with modifications [MSA1 were commercially synthesized to optimize the codon usage for expression in E. coli, and then ligated into the EcoRI-SalI site of pET28a and the SacI-XhoI site of pET32a, respectively. The coding sequence of E. coli SHMT gene was PCR amplified from genomic DNA of E. coli strain Top10 using the primer sequences listed in EcoRI-SalI site of pET32a. After confirmation by sequencing the correct constructs were transformed into GS245(DE3)pLysS. Three independent clones of each GS245(DE3)pLysS strain carrying different plasmids were grown in Lysogeny broth (LB) medium overnight at 37°C. Cells were collected by centrifugation and washed twice with M9 minimal medium , 0.4% w/v glucose, 50 μg ml-1 phenylalanine, 10 μg ml-1 thiamine, 2 mM MgSO4, 0.1 mM CaCl2, 5 μM FeSO4, 3 nM (NH4)6Mo7O24, 400 nM H3BO3, 30 nM CoCl2, 10 nM CuSO4, 80 nM MnCl2, and 10 nM ZnSO4). Cells were re-suspended and diluted in M9 minimal medium to an equal optical density at 630nm of 0.5. Ten micro litres of cells were transferred to 1 mL of M9 minimal medium with required antibiotics in a 96-well 2 mL deep plate. Different concentrations of IPTG were added for induction. 96-well plates were placed in an orbital shaker and incubated at 37°C and 350 rpm. The OD630 value was measured after 24 h incubation using a plate reader.The complementation of an ications , 73. Themsa1-1 using a DNeasy Plant Mini Kit (Qiagen). DNA samples were sent to Beijing Genomics Institute at Shenzhen  for bisulfite treatment, library construction and high throughput sequencing. Bisulfite treatment was carried out by an EZ DNA Methylation-Gold kit (ZYMO Research). For data analysis, the adapter sequences were trimmed and low-quality reads (q < 20) were removed. Clean reads were mapped to the A. thaliana genome (TAIR10) using BSMAP aligner [Plants used for whole-genome bisulfite sequencing were grown on MGRL media for two weeks. Genomic DNA was extracted from shoots and roots of WT or  aligner  with reaP-values calculated from the test were then adjusted using the Benjamini-Hochberg method to control the false discovery rate (FDR). Windows with adjusted P-value ≤ 0.05, fold-change of methylation level between two samples > 2 and coverage > 0.6 was considered as a DMR. Overlapped windows was merged into DMRs and filtered to include only those with minimum 200 bp length. Genes with at least one DMR in the gene body, 2 kb upstream, or 2 kb downstream flanking regions were considered as differentially methylated genes (DMGs). Gene ontology (GO) enrichment analysis was performed on hyper- and hypo-DMGs of shoots and roots, and the overlapped DMGs using DAVID [A sliding-windows approach with five CGs (CHGs or CHHs) as a window at one C interval was used to identify the DMRs. DMRs were identified by comparing the methylation level at CG, CHG and CHH contexts separately. A Fisher’s exact test was used to determine the DMR between samples. ng DAVID . Only thmsa1-1 plants were grown on MGRL media with 1500 μM (+S) or without (-S) sulphate for two weeks. The genomic DNA was extracted from shoots and roots using a DNeasy Plant Mini Kit (Qiagen). One microgram genomic DNA was mixed with restriction buffer and then split into 2 × 50 μL with either 2 μL McrBC enzyme (New England Biolabs) or 2 μL 2% (v/v) glycerol as nondigested control. After overnight digestion and deactivation, a standard PCR was performed using the primers to amplify the promoter sequence of SULTR1;1. EF-1α (AT5G60390) was used as a negative control. The primer sequences used for PCR are listed in WT and http://swissmodel.expasy.org) [http://www.expasy.org/spdbv/) [Homology modelling was conducted using the web-based SWISS-MODEL platform (asy.org) . The cryasy.org)  with bou/spdbv/) . The MUTS1 Fign = 6). Single and double asterisks indicate values significantly different between WT and msa1-1 mutant at P ≤ 0.05 and P ≤ 0.01, respectively (Student’s t test). DW, dry weight.Plants were grown on MGRL agar media without sulphate (S0), with 15 μM (S15), 1500 μM (S1500) or 4500 μM (S4500) sulphate for two weeks. Total S in both shoots (A) and root (B) were determined. Data are presented as means ± SD ((TIF)Click here for additional data file.S2 Figer-0, msa1-1 and msa1-1 X Ler-0 F1 plants. Data are presented as means ± SD (n = 12). Columns with different letters indicate significant difference . DW, dry weight.  The frequency distribution of leaf S and Se content in msa1-1 X Ler-0 F2 population. ICP-MS data is accessible using the digital object identifier (DOI) 10.4231/T99G5JRP (see http://dx.doi.org/). S and Se content in the leaves of WT Col-0, L(TIF)Click here for additional data file.S3 FigA. thaliana, rice, human, rabbit, mouse, yeast and E. coli. Sequence alignment was performed by using Clustal W. Identical and similar residues are displayed in black or grey background. The mutated amino acid in MSA1 of the MSA1 mutant was marked with red box. The binding sites of pyridoxal-5’-phosphate (PLP) and folate were marked with close red triangle and open blue triangle, respectively, based on the crystal structure of rabbit SHMT1 (OcSHMT1). The nuclear localization signal (NLS) of MSA1 was marked with magenta box. Protein sequences were extracted from GenBank. Arabidopsis : AtSHM1 (At4g37930), AtSHM2 (At5g26780.1), AtSHM3 (At4g32520), AtSHM4 (At4g13930), AtSHM5 (At4g13890), AtSHM6 (At1g22020), AtSHM7/AtMSA1 (At1g36370); Rice (Oryza sativa): OsSHM1 (Os03g0738400), OsSHM2 (AK101450), OsSHM3 (Os12g0409000), OsSHM5 (Os01g0874900), OsSHM6 (Os05g0429000); Human (Homo sapiens): HsSHMT1 (AAH07979.1), HsSHMT2 (AAH11911.1); Rabbit (Oryctolagus cuniculus): OcSHMT1 (P07511), OcSHMT2 (NP_001075874.1); Mouse (Mus musculus): MmSHMT1 (AAH26055.1), MmSHMT2 (AAH04825.1); Yeast (Saccharomyces cerevisiae): ScSHMT1 (P37292), ScSHMT2 (P37291); E. coli (Escherichia coli): EcSHMT (ACI79831.1). (B) Homology model of MSA1 generated by using rabbit SHMT1 (PDB ID: 1ls3) as a template. The Ribbon diagram only showed the residues from 136 to 591 of MSA1 with good alignment with the template. Black dash square indicated the region with mutated residue Ser186 in msa1-1 mutant. (C) Close-up structure of the region with Ser186 and its neighbour residues in wild type MSA1 protein. (D) Close-up structure of the same region as in (B) with Ser186 mutation to Phe. The binding sites of pyridoxal-5’-phosphate (PLP) and folate were shown in magenta and blue, respectively, in (B) to (D). The Ser186 and Phe are shown in red. The green dash lines in (E) and (F) indicate H-bonds, and purple dash lines in (D) indicate steric hindrance.(A) Amino acid sequence alignment of SHM proteins from (TIF)Click here for additional data file.S4 FigMSA1. Blue bars, grey lines and white bars represent exons, introns and untranslated region, respectively. Point mutation site was indicated by vertical lines. T-DNA insertion sites were indicated by triangles. The primers using for genotyping and qRT-PCR were shown as black arrows. (B) Genotyping T-DNA insertion lines. Gene specific primers (FP and RP) and left border primer of the T-DNA insertion (LB) were used. (C) Quantification of expression of MSA1 in two T-DNA lines by qRT-PCR. The expression level was normalized to the internal control gene UBQ10, and relative expression level was presented as 2-ΔΔCt with WT as reference. Data were shown as means ± SD (n = 3).  S and Se content in the leaf of WT, msa1-1 and T-DNA insertion lines. Data are presented as means ± SD (n = 11 or 12). Columns with different letters indicate significant difference . DW, dry weight.(A) Gene structure of (TIF)Click here for additional data file.S5 Fig(A) Nuclear localization of MSA1 was not affected by S deficiency. Transgenic plants harboring wild-type MSA1 (GFP-MSA1w) and mutated MSA1 (GFP-MSA1m) were grown on S sufficient media  or S deficient media . The nucleus was stained by DAPI. Bar, 10μm. (B) Subcellular localization of MSA1 in the leaf. Transgenic plants harboring wild-type MSA1 (GFP-MSA1w) and mutated MSA1 (GFP-MSA1m) were grown on half-strength MS media for 5 days. The nucleus were stained by DAPI. Auto fluorescence indicates chloroplast. Bar, 10μm. (TIF)Click here for additional data file.S6 Fig14C-labeled Ser and monoglutamylated THF as substrates. pCal-n and pHUE are the empty vectors for expressing SHM1 and 2, and MSA1, respectively. (B) MSA1 failed to complement an E. coli glycine auxotroph (GS245(DE3)pLysS; shmt-). Growth of various strains in liquid media supplemented with different concentrations of IPTG for induction. 50 μg mL-1 glycine was added as control. OD630 was measured after 24 hours incubation. Bacteria SHMT was used as the positive control. Data in (A) and (B) are presented as means ± SD (n = 3). Columns with different letters in (A) indicate significant difference .(A) The SHM activity of purified recombinant SHM proteins. SHM activity was determined using (TIF)Click here for additional data file.S7 FigDNA methylation level was calculated by the density of methylated C in each 100 kb windows and the highest density windows in each contexts was designated as 100%.(TIF)Click here for additional data file.S8 Figmsa1-1.Numbers of overlapping DMRs were shown separately in hyper-methylated in shoots and hypo-methylated in roots of WT and (TIF)Click here for additional data file.S9 Figmsa1-1 mutant grown in soil for five weeks and the transcript level was determined by quantitative RT-PCR. The expression level was normalized to the internal control gene UBQ10, and presented as 2^(deltaCt) with means ± SD (n = 3). Single and double asterisks indicate values significantly different between WT and msa1-1 mutant at P ≤ 0.05 and P ≤ 0.01, respectively (Student’s t test).Total RNA was isolated from the shoot and root of WT and (PDF)Click here for additional data file.S10 Figmsa1-1 mutant. 08620LP2 and RP2 are SULTR1;1-specific primers, 78000LP1 and RP1 are SULTR1;2-specific primers, and XR2 and JL202 are T-DNA border primers. M, DNA ladder. The primer sequences are listed in The dCAPS primer 78730SNP2 was used to genotype the point mutation in (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 TableThe position highlighted with blue and red colours indicate the location of the shoot and root DMRs, respectively. The position highlighted with grey colour indicate the location of primers used for the chop-PCR in (XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.