CRYAA were associated with susceptibility to ARC in a Han Chinese population and to clarify the mechanism of this association.Alpha A-crystallin (CRYAA) is considered critical for the maintenance of lens transparency and is related to the pathogenesis of age-related cataracts (ARCs), especially the nuclear subtype. As the 5′ untranslated region (5′ UTR) modulates gene expression, the purpose of current study was to investigate whether single nucleotide polymorphisms (SNPs) in the 5′ UTR of CRYAA were identified in 243 nuclear ARC patients and 263 controls using polymerase chain reaction and DNA sequencing. Allele and genotype frequencies were calculated and compared between two groups. Haploview 4.2 was used to calculate the linkage disequilibrium index, and the SHEsis analysis platform was used to infer haplotype construction. A dual-luciferase reporter gene assay was used for transcription of CRYAA in the presence of a protective haplotype with individual SNP alteration, Chromatin immunoprecipitation (ChIP) was employed to determine whether SNPs regulated CRYAA expression by altering the binding affinity of transcription factors.SNPs in the 5′ UTR (−1 to −1000) of P = 0.000357, odds ratio [OR] = 1.837), rs13053109 , and rs7278468 . The haplotype C-G-T  increased the risk of nuclear ARC, whereas the haplotype T-G-G  decreased the risk. The haplotype C-G-T decreased CRYAA transcription through rs7278468, which is located in the binding site of specificity protein 1 (Sp1). Furthermore, the G allele of rs7278468 increased CRYAA transcription by enhancing the binding affinity of Sp1.Three polymorphisms were identified in the 5′ UTR of CRYAA: rs3761381  contains supplementary material, which is available to authorized users. Age-related cataracts (ARCs), which are a major cause of blindness worldwide, are characterized by lens opacities and visual impairment due to degenerative changes in the lens in the elderly . The cauAs a major structural protein component expressed in the lens, alpha A-crystallin (CRYAA) is considered critical for the maintenance of lens transparency . Many stSLC16A12 gene were involved in the pathogenic consequences of ARC [CRYAA contribute to ARC susceptibility in a Han Chinese population, especially the nuclear subtype (the most frequent form).Previous research demonstrated that the 5′ untranslated region (5′ UTR) acted as a regulatory element in genes and that it was associated with modulating the expression of gene-coding regions . Single s of ARC . Recentls of ARC . HoweverThe present study focuses on polymorphisms within the 5′ UTR of CRYAA (−1 to −1000) and attempts to shed light on the development of nuclear ARC in a Han Chinese population.Subjects were recruited from the Eye and ENT Hospital of Fudan University. All the subjects underwent a full ophthalmic examination, including visual acuity, slit-lamp microscopic examinations, and ophthalmoscopic examinations. There was no consanguinity between the subjects . All the enrolled subjects self-identified as Han Chinese . This research was approved by the Institutional Review Board and followed the tenets of the Declaration of Helsinki. All the subjects signed informed consent forms.P ≥ 2), or mixed type .After pupil dilation with 1% tropicamide, a trained ophthalmologist graded the lens opacity of each right eye according to the Lens Opacity Classification System (LOCS) III. Opacity was classified as nuclear opalescence (NO), nuclear color (NC), cortical (C), and posterior subcapsular (P). The grading consisted of six standards for NO and NC (standards 1 to 6) and five standards for C and P (standards 1 to 5). Each standard was assigned using decimals to interpolate between the reference standards, with the assigned scores ranging from 0.1 to 6.9 for NO and NC and 0.1 to 5.9 for C and P. The subtype of the cataract was then classified as nuclear (NO or NC ≥3), cortical (C ≥ 2), posterior subcapsular cataract (PSC)  subjects younger than 45 years; (2) pseudophakia or aphakia in either eye; (3) the presence of other eye diseases, such as dislocated lens, trauma, uveitis, high myopia, glaucoma, macular diseases, and retinal detachment; (4) previous ocular surgery in either eye; and (5) a history of diabetes, kidney disease, respiratory disease, cancer, or tumors.Five milliliters of peripheral blood samples were collected in EDTA tubes from all the subjects was and stored at −80 °C until use. DNA was isolated from whole blood cells using a Mammal Blood Genomic DNA Extraction Kit , following the manufacturer’s protocol, and was stored at −20 °C until used for genotyping.CRYAA, the polymerase chain reaction (PCR) and DNA direct sequencing were performed. A set of primers (forward: GGTGACACAGCAAGACTCCA and reverse: CACGTCCATGTTCAGCTTTG) from Generay Biotechnology Co., Ltd.  was used to amplify the target fragment. The PCR reactions were performed in 50 μl reaction mixtures, consisting of 25 μl of PrimeSTAR Max Premix (Takara), 20 μl of RNase-free water, 1 μl of the forward primer, 1 μl of the reverse primer, and 1 μl of extracted genomic DNA. The PCR program included a 94 °C activation step for 3 min, followed by 30 cycles of 94 °C for 20 s, 60 °C for 20 s, 72 °C for 40 s, and 72 °C for 65 s. The PCR products were sequenced using ABI 3730xl .For analyzing the polymorphism in the 5′ UTR (−1 to −1000) of The human lens epithelium (HLE) B3 cell line obtained from American Type Culture Collection  was cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 20% fetal bovine serum (FBS). Before transfection, the cells were seeded in six-well plates and grown overnight, so that the cell density reached approximately 70%. A mixture of 2 μg of plasmid DNA and 4 μl of Lipofectamine™ 2000 (Invitrogen) reagent in 2 ml of serum-free medium was then added in a six-well plate. The cells were collected for luciferase activity testing and chromatin immunoprecipitation (ChIP) analysis 72 h and 48 h later, respectively.CRYAA 5′ UTR (−1 to −1000) was amplified using primers, as described above and then inserted into the PGL3-Basic vector (Invitrogen) using the restriction enzymes Hind III and KpnI (Takara). The CRYAA 5′ UTR containing each SNP mutation was constructed using site–directed mutagenesis, with the following primers: rs3761381-mut forward: ATCATGTGGGTGGTGGGTCT, reverse: ATGCCTTGATGTAACATCCCC; rs13053109-mut forward: GGTGAGACTCTGAGGACGATGTGT, reverse: ACACATCGTCCTCAGAGTCTCACC; rs7278468-mut forward: GGGTGTGTGCTCTCCCTCCTCT, reverse: AGAGGAGGGAGAGCACACACCC.The human The HLE cells were cultured in six-well plates with 2 μg of either PGL3-Basic-CRYAA 5′ UTR or PGL3-Basic plasmid and 20 ng of pRL-TK and then transfected into cells, as described above. Then, 72 h after transfection, luciferase activities were checked by a Dual-luciferase Reporter (DLR™) Assay System (Promega), according to the manufacture’s protocol.The transcription factor binding sites of the identified SNPs of CRYAA 5’UTR were predicted by MEME SUITE as described previously .CRYAA 5′ UTR was conducted 48 h after transfection using a Pierce Agarose ChIP Kit (Thermo Scientific), according to the manufacture’s protocol. The antibodies used for ChIP included anti-human IgG (Abcam) and anti-specificity protein 1 (Sp1) (Abcam). The captured genomic DNA fragments were then purified with Premix Taq™ (Takara). The primers used for the ChIP PCR were as follows: CRYAA ChIP forward: CTGAGGACGATGTGTCTAACCTC, reverse: AGGCCTGGACTCAGCTGA. CRYAA ChIP NC: forward: ACCCTGACAGGAGCAGCCCA, reverse: TCCTCCAGGGGTCACATGC. The ChIP-quantitative PCR (qPCR) data relative to Sp1 were calculated using the 2-ΔΔCt methods and presented as % input.ChIP analysis of HLE cells transfected with a plasmid containing the t-test or Fisher’s chi-squared test, depending on the variables types. SPSS for Windows, version 17.0  was used in the statistical analysis of the above data and for the calculation of odds ratios (ORs). The Hardy–Weinberg equilibrium (HWE), linkage disequilibrium (LD) and the haplotype analysis was conducted using the SHEsis software platform [P < 0.05 was considered statistically significant.Differences between the values were evaluated using a two-tailed Student’s platform . The expp = 0.667).In total, 243 unrelated ARC patients and 263 control subjects were included in this study. The mean ages of the ARC patients and controls were 70.06 ± 6.22 years and 69.01 ± 4.64 years, respectively (P > 0.05). The frequencies of the rs3761381 T allele  and rs7278468 G allele  were significantly higher in the control group compared to the ARC group. With regard to rs13053109, there were no significant differences in the genotype or allele frequency between the ARC patients and the controls.Three polymorphisms were identified in the 5′ UTR of CRYAA: rs3761381 (C > T), rs13053109 (G > C), and rs7278468 (T > G) , whereas the haplotype T-G-G  seemed to confer protection against ARCs.The D’ values for the LD of the three SNPs are shown in Fig. Based on the position of these three SNPs, we constructed luciferase reporter vectors containing the CRYAA 5′ UTR and either T-G-G (protective haplotype) or C-G-T (risk haplotype) and evaluated whether they could influence the transcriptional activity of the gene. Seventy-two hours after transfection of the HLE cells, a dual-luciferase reporter assay showed that the luciferase activity of the cells transfected with the vector containing the CRYAA 5′ UTR Fig.  was moreCRYAA 5′ UTR with the T-C-T haplotype decreased approximately 11% (P < 0.05) compared with that of the protective T-G-G haplotype. These data indicated that the allelic change in rs7278468 affected CRYAA expression.To distinguish which individual SNPs of the haplotypes in the CRYAA 5’ UTR were predominantly responsible for the regulation of transcriptional activity, site-directed mutagenesis was employed to alter the single base of each individual SNP present in the protective T-G-G haplotype, one by one. Subsequent transcriptional activity was then analyzed. As shown by a dual-luciferase reporter assay, 72 h after transfection, the transcriptional activities of the 5’ UTR containing the individual alleles of rs3761381 and rs13053109 occurring as a single base variation were not significantly different from those of the protective T-G-G haplotype Fig. . HoweverCRYAA transcriptional activity, as it lies upstream of the transcription start site. It is feasible that it might influence the binding affinity of one or more transcription factors and affect the transcriptional activity of CRYAA. Thus, using the MEME SUITE, transcription factor binding prediction of the sequence around rs7278468 was performed. The rs7278468 T allele is located in the binding motifs of several transcription factors, including specificity protein 1 (Sp1)  in a northern Italian population [This study demonstrated evidence of the involvement of alleles, genotypes, and haplotypes of SNPs in the 5′ UTR of pulation . Howeverpulation –19. The pulation , 21. FurAs a molecular chaperone, CRYAA protects other crystallins from aggregation or inactivation and to traps aggregation-prone denatured proteins, which are suggested to delay the progression of ARC . StudiesSLC16A12 gene affected the pathogenic consequences of ARC [CRYAA via binding to the binding motif in the 5′ UTR of CRYAA, with an increase in the binding strength of the Sp1 binding motif in the rs7278468 G allele increasing CRYAA transcription. The resulting increased in CRYAA transcription seemed to enhance the expression of the alpha A-crystallin protein in the lens, making individuals with the rs7278468 G allele invulnerable to the development of nuclear ARC.SNPs in the 5′ UTR play a critical role in gene expression and are involved in the pathophysiology of disease. Seshadri identified a SNP, rs366316, in the 5′ UTR of CD1a that was strongly associated with CD1a expression . SNPs ids of ARC . As showSp1 is a ubiquitously expressed transcription factor, which plays a critical role in regulating plenty of genes required for normal cell function , 25. In To address the limitation of the sample size, larger sample studies are required to validate the association we have observed. Further studies could also be focused on the association of SNPs with cortical and PSC cataracts.CRYAA transcription, rs7278468 appeared to be mainly responsible for the alteration in the transcriptional activity of the 5′ UTR of CRYAA. It exerted this effect through its G allele, which strengthened the binding affinity of Sp1. Manipulating these polymorphisms may provide a strategy to prevent or slow the progression of ARC.This study identified three SNPs and their haplotypes in the 5′ UTR of CRYAA that appeared to be associated with nuclear ARC. As shown by the analysis of the effects of the individual SNP alleles on Additional file 1:The transcription factor binding sites overlapping rs7278468. This is the raw data of the in-silico analysis. (DOCX 52 kb)Additional file 2: Figures S1 and S2.The whole gels of the NC and Sp1 ChIPs respectively. This is the raw data of the chromatin immunoprecipitation (ChIP) analysis described above. (DOCX 645 kb)


Alternative cleavage and polyadenylation (ApA) is known to alter untranslated region (3ʹUTR) length but can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the coding region. We analyzed 46 3ʹ-seq and RNA-seq profiles from normal human tissues, primary immune cells, and multiple myeloma (MM) samples and created an atlas of 4927 high-confidence IpA events represented in these cell types. IpA isoforms are widely expressed in immune cells, differentially used during B-cell development or in different cellular environments, and can generate truncated proteins lacking C-terminal functional domains. This can mimic ectodomain shedding through loss of transmembrane domains or alter the binding specificity of proteins with DNA-binding or protein–protein interaction domains. MM cells display a striking loss of IpA isoforms expressed in plasma cells, associated with shorter progression-free survival and impacting key genes in MM biology and response to lenalidomide. Recognition of intronic polyadenylation (IpA) signals can lead to expression of truncated proteins lacking C terminal domains. Analysis of 3ʹ -seq and RNA-seq shows that IpA is widespread in circulating immune cells, while multiple myeloma cells show loss of IpA isoforms that are normally expressed in plasma cells, impacting key genes in the disease. For example, analysis of a human ApA tissue atlas established that half of human genes express multiple 3ʹUTRs, enabling tissue-specific post-transcriptional regulation of ubiquitously expressed genes1. However, ApA events can also occur in introns rather than 3ʹUTRs, generating either non-coding transcripts or transcripts with truncated coding regions that lead to loss of C-terminal domains in the protein product.Alternative cleavage and polyadenylation (ApA) is generally viewed as the selection of ApA signals in the 3ʹ untranslated region (3ʹUTR), leading to the expression of different 3ʹUTR isoforms that code for the same protein. Recent computational analyses of 3ʹ-end sequencing data have characterized the nature and extent of ApA in mammalian 3ʹUTRsIGHM) locus9. In mature B cells, recognition of the polyadenylation signal in the 3ʹUTR produces the full-length message, including two terminal exons that encode the transmembrane domain of the plasma membrane-bound form of immunoglobulin M  signal occurs in the immunoglobulin M heavy chain  when splicing outcompetes polyadenylation and otherwise producing a truncated mRNA16. As the defining event is the recognition of an IpA signal, we call these transcripts IpA isoforms. It is now possible to recognize the widespread expression of IpA isoforms through the analysis of 3ʹ-end sequencing data.In the splicing literature, isoforms generated through recognition of an IpA signal are often described as ‘alternative last exon’ events1. Through integration with RNA-seq profiles in B-lineage and MM cells as well as external data sets and annotation databases, we assembled an atlas of confident IpA isoforms supported either by independent data sources or very highly expressed in at least one cell type. We found that IpA isoforms are widely expressed, most prevalently in blood-derived immune cells, and that generation of IpA isoforms is regulated during B-cell development, between cellular environments, and in cancer. IpA events in immune cells are enriched at the start of the transcription unit, leading to IpA isoforms that retain none or little of the coding region (CDR) and hence represent a class of robustly expressed non-coding transcripts. IpA events that occur later in transcription units can lead to truncated proteins often lacking repeated C-terminal functional domains and thus contribute to the diversification of the proteome.We identified robust ApA events that occur in introns and quantified IpA isoform expression using 3ʹ-seq across human tissues, immune cells, and in multiple myeloma (MM) patient samples. We focused on immune cells because it is feasible to obtain pure populations of primary cells and because B cells expressed the largest number of IpA isoforms in our previous tissue atlas1 and combined it with a newly generated data set from normal and malignant primary immune cells. The new immune cell profiles (n = 29) were all performed with biological replicates and included lymphoid tissue-derived naive B cells (NB), memory B cells (MemB), germinal center B cells (GCB) and CD5+ B cells (CD5+B), blood T cells and plasma cells (PCs), and MM derived from bone marrow aspirates , cell types (embryonic stem (ES) cells, naive B cells from peripheral blood (blood NB)), and cell linesiles n = 9 were alIGHM is highly expressed in PCs while the full-length transcript, encoding membrane-bound IgM, is the dominant isoform in NB cells  of IpA events from our 3ʹ-seq peak calls -adjusted P < 0.1) versus almost no significant differences at randomly chosen positions in introns . Finally, events with high expression in at least one cell type were also included (323 events). 13% (n = 743) of IpA events could not be validated by any of these criteria and thus were excluded from further analysis , a resource based on 3ʹREADS data18, and 72% (3581/4927) have support from existing PolyA-seq, as they do not include the immune cell types that we profile here. Therefore, while our atlas does not exhaustively sample human tissues, we have assembled the most comprehensive IpA resource to date.To assemble an atlas of confident IpA events, we compared each intronic 3ʹ-seq peak against external annotation and data sources  of 3.71–3.83 for IpA isoforms in PCs, blood NB, and blood T versus log2 TPM of 4.53–5.15 for full-length transcripts; Fig. IpA isoforms are robustly expressed, with median expression of the same order of magnitude as for full-length isoforms (logn = 3365), and almost all of these in at least two immune cell types. Non-immune tissues like testis and ES cells express tissue-specific IpA isoforms, but the majority are produced from tissue-specific genes.Figure To begin to assess the impact of IpA isoform expression, we computed the fraction of retained CDR for each IpA isoform relative to the full-length annotated CDR. The histogram of retained CDR fraction for atlas events showed a uniform distribution except for a substantial overrepresentation of IpA isoforms that lose all or almost all of the CDR Fig. . Howeverr = −0.86, Fig. r = 0.60, Fig. We observed a significant negative correlation across tissues between the frequency of IpA isoform expression and length of retained CDR  is not recognized and the entire sequence from this donor splice site to the intronic cleavage site is included in the isoform; and ‘skipped terminal exon’, where the IpA isoform introduces a new small exon ending at the intronic cleavage site, requiring recognition of the previous donor splice site and a new acceptor splice site , although the effect sizes were more modest. However, IpA genes have high AT content compared to the genes lacking IpA events, so AT content is enough to segregate IpA genes from non-IpA genes . Therefore, genomic architecture and sequence composition may facilitate IpA isoform expression, but high background AT content is also strongly associated with IpA genes.Previously, U1 small nuclear ribonucleoprotein (snRNP) expression and the presence of U1 snRNP motifs early in the transcription unit were found to play a crucial role in preventing premature cleavage and polyadenylationrms Fig. . To contrms Fig. . We agai25. A majority of expressed IpA isoforms significantly differed in usage between NB cells from lymphoid tissue and blood T cells . We also found differential IpA usage between B-lineage cell types, with PCs in particular showing strikingly increased IpA site usage compared to tissue-derived NB cells  approach to determine significant changes in the relative expression of IpA isoforms compared to full-length transcripts (usage of IpA) across normal immune cells (see Methods)lls Fig. . Howeverlls Fig. . Thus, Ills Fig. 26.Fig. 3P < 10−5). However, the decrease in full-length isoform expression was modest, indicating that IpA usage does not predominantly result in a ‘switch-like’ change between full-length and IpA isoform expression.Figure NFKBID has six ankyrin domains, while the IpA-generated truncated protein retains four of them. Similarly, the full-length protein of the transcription factor PATZ1 has seven zinc-finger domains, while different IpA isoforms are predicted to encode either four or five zinc-fingers , IpA results in a preferential loss of DNA-binding or PPI domains but avoids the loss of active sites  and are significantly depleted among IpA genes Fig. . Neverth27. Therefore, they likely generate micropeptides or represent non-coding RNAs , which are typically enriched in the 3ʹUTRs of coding transcripts, but not for RNA-binding proteins usually bound to introns, supporting the exonic nature of the predicted non-coding transcripts.A large fraction of IpA isoforms that are differentially used among normal immune cell types are in fact 5ʹIpA isoforms (487 out of 1281). Through de novo RNA-seq assembly, we were able to resolve the transcript structure for 954 of the 5ʹIpA isoforms (see Methods) and found that 469 of these have low predicted coding potential with open reading frames (ORFs) encoding fewer than 100 amino acids33, we investigated whether IpA is also dysregulated in cancer. Since PCs express the highest number of IpA isoforms among the tissue-derived B cells, we compared IpA isoform expression between normal and malignant PCs, derived from MM patients (n = 15). As MM is a heterogeneous disease, we used hierarchical clustering based on IpA isoform expression to define three patient subgroups . The significant events in patient group 1 largely represent a superset of those in group 2 . While RNA-seq signal is often not an accurate readout of IpA isoform expression levels  to use as a signature for the extent of IpA loss in the larger RNA-seq cohort.To investigate whether IpA isoform expression is correlated with any clinical factor, we examined a cohort of 286 MM patients with RNA-seq profiles and clinical information from a separate studyn = 126) with low median IpA usage and group B (n = 160) with high median IpA. Interestingly, group B patients, whose IpA usage is more similar to normal PCs, have improved progression-free survival (P < 0.05) compared to group A patients  to remove patients whose neighbors had inconsistent cluster assignment. This improved the contrast between low IpA group A-filtered (n = 64) and high IpA group B-filtered patients -mediated degradation of IKZF1 by lenalidomide35. The IpA isoform of CUL4A is translated into a truncated protein (1–174 amino acids) that retains only its N-terminal domain  has been shown to act in a dominant negative manner45. The CUL4A-IpA isoform retains its N-terminal domain and hence the potential to interact with DDB1  C and U2AF65, involved in the regulation of Alu exonization, a mechanism known to control the expression of intronic exons51. The fact that more prevalent use of IpA signals, shorter IpA isoforms, and shorter 3ʹUTRs are all correlated across tissues suggests that the abundance of the same global co-transcriptional factors may be partially responsible for all three properties.IpA isoform expression is thought to be regulated by competition between splicing and cleavage-polyadenylation reactions53. The tendency to retain certain introns may provide the polyadenylation machinery time to recognize IpA signals for cleavage and 3ʹ end processing. Interestingly, we did find a statistically significant co-occurrence of introns with IpA events and retained introns ; potentially, these non-coding RNAs serve as scaffolds for RBPs and thereby exert a regulatory role in trans on other RNAs.A surprising finding was the enrichment of IpA isoforms at the 5ʹ end of the transcription unit in immune cells. We identified 469 5ʹIpA isoforms with predicted coding sequence generating fewer than 100 amino acids. These IpA isoforms are either non-coding or represent a source of micropeptidesn = 2667), however, are predicted to generate truncated proteins that retain at least one domain and have the potential to be functional. Notably, IpA genes encode larger proteins that contain significantly more domains than proteins generated from non-IpA genes. Strikingly, proteins encoded by IpA genes are enriched for repeated protein domains, which in a majority of cases are only partially lost, thus modulating but not abolishing overall protein function. Moreover, the active sites of enzymes are generally retained in IpA-generated truncated proteins, again resulting in proteins with similar function as the full-length proteins but different affinity or different binding partners. Thus, the cell type-specific expression of truncated proteins generated through IpA may be a widely used mechanism to diversify the proteome, not a peculiarity of a few well-known examples like IgM.The majority of IpA isoforms , L-selectin, transforming growth factor-α, or CD40IKZF1, the gene encoding IKAROS, a transcription factor whose full-length form is predominantly expressed in mature B cells. The activity of IKZF1 is known to be essential for the development of B-cell precursors62, but its DNA-binding activity appears to be reduced in PCs through a switch to expression of the apparently non-DNA-binding IpA isoform.It is interesting to speculate why immune cells generate more IpA isoforms than solid tissues. One possibility is that circulating immune cells are specialized for secretion of cytokines, and IpA provides a mechanism for generation of soluble isoforms of proteins whose full-length isoforms are membrane bound. In addition, the immune system requires repeated execution of complex differentiation programs. Perhaps immune cells have acquired IpA events for specialization of transcriptional and post-transcriptional programs during cellular differentiation through partial loss of repeated DNA-binding and RBP domains. An important example is IZKF1 displays a switch-like loss of IpA isoform expression and rescue of full-length transcript expression in MM, providing a key therapeutic target through Cereblon-mediated degradation by lenalidomide35. Intriguingly, some MM patients retain expression of PC IpA isoforms that may be therapeutically relevant, such as CUL4A-IpA. CUL4A acts in the DDB1-CUL4A-ROC1 E3 ubiquitin ligase complex that interacts with Cereblon to ubiquitinate and degrade IKZF1. CUL4A-IpA may act as a dominant negative, able to bind DDB1 but unable to recruit the E2 enzyme required for substrate ubiquitination. In patients who retain expression of CUL4A-IpA, although displaying similar IpA usage to PCs overall, we hypothesize that this truncated protein isoform may provide a mechanism of resistance to lenalidomide. Interestingly, not all cancer cells show depletion of IpA isoforms, as we found increased IpA isoform expression in another B-cell malignancy, chronic lymphocytic leukemia, reported elsewhere.Indeed, IpA isoform expression changes in different stages of B-cell development and after environmental changes and importantly is dysregulated in cancer. A majority of the MM patients that we profiled showed a striking loss of IpA isoforms normally expressed in PCs. As a group, genes that lose IpA expression in MM samples compared to PCs also upregulate full-length transcript expression, presumably rescuing the function of the full-length protein to varying degrees. Here, Normal B-cell populations derived from tissues were obtained from lymphadenectomies performed at Weill Cornell Medical Center, NY. Blood immune cells were obtained using buffy coats obtained from the New York Blood Center. Mononuclear cells were obtained using Ficoll centrifugation. After that, cells were prepared for fluorescence-activated cell sorting (FACS) to obtain pure populations. Cells were washed with ice-cold phosphate-buffered saline (PBS) once, incubated with appropriate fluorochrome-conjugated antibodies for 30 min at 4 °C and washed twice with ice-cold PBS containing 0.5% fetal calf serum. The following antibodies were used for FACS: anti-CD3-PE , anti-CD5-FITC , anti-CD14-PECy7 , anti-CD19-APC , anti-CD27-PE , anti-CD38-APC , and anti-CD38-PE . The purity of immune cell populations was analyzed by FACS and the cells were immediately dissolved in TRI Reagent (Ambion) for RNA extraction, followed by 3ʹ-seq or RNA-seq library preparation. Primary MM cells or PCs were isolated using Ficoll-Hypaque density gradient sedimentation from bone marrow aspirates of MM patients or healthy individuals respectively followed by anti-CD138 microbeads  selection, in accordance with the Declaration of Helsinki following informed consent and Institutional Review Board (Dana-Farber Cancer Institute) approval.63, identification, and quantification of peaks were performed as described by Lianoglou et al.1. The peaks were assigned to genes using RefSeq annotations. To obtain an atlas of robust cleavage events in 3ʹUTRs and introns, we started with all the peaks that were detected by peak calling of all the pooled samples and then followed a series of steps to filter lowly expressed peaks and the ones that potentially originate from different artifacts.Preprocessing of 3ʹ-seq libraries, read alignment (hg19)n = 2841; 0.16%) (https://sites.google.com/site/anshulkundaje/projects/blacklists)64; (ii) internally primed peaks1 ; and (iii) antisense peaks .Removing artifacts: The peaks potentially resulting from different artifacts were identified and removed: (i) peaks overlapping blacklisted regions of human genome (n = 11) overlapping with parts of the genome coding for immunoglobulins were removed. Even after this correction, one sample of plasma cells had a high number of intergenic reads (PC2). Thus, this sample was not used for identification of robustly expressed isoforms but only to quantify them. The library size was reduced accordingly for all samples, since the peaks described above result either from sequencing artifacts or from skewed expression of specific genomic regions.Removing the immunoglobin peaks: Our data set included plasma cells which are fully differentiated B cells that secrete antibodies. As plasmas cells produce massive quantities of antibodies, a large fraction of 3ʹ-seq reads mapped to immunoglobulin loci on chromosomes 2 and 14. It was essential to account for this skewed expression of specific genomic regions in order to get a reasonable quantification for the expression of other genes. Thus, peaks (n = 336) were removed from further analysis. This resulted in the removal of 8437 peaks from the atlas. Since we were interested in investigating the IpA isoforms of protein coding genes, peaks falling in introns that potentially originated from microRNAs, small nucleolar RNAs, and retrotransposons were also removed (n = 4722). Genes that were on the opposite strand but had a 3ʹUTR end in the intron (100 nt) of a convergent gene can create artifactual antisense peaks in the intron. Thus, peaks in introns that were close to the end of an opposite strand 3ʹUTR were also removed (n = 2091). This corresponded to discarding peaks in the introns of 630 genes. There are genes where the end of the 3ʹUTR might fall in the intron of the downstream gene on the same strand. This would also create peaks in introns that are contributed by the preceding gene. Therefore, peaks in the intron that were within 5000 nt of the 3ʹ end of the 3ʹUTR of the previous gene were discarded (n = 2079); the discarded peaks came from the introns of 785 genes.Removing ambiguous peaks: Some genes in the genome overlap with each other. In such cases, it is difficult to assign 3ʹ-seq reads to the genes accurately, and thus such genes , and therefore their usage was calculated relative to the total expression of both IpA isoforms and 3ʹUTR isoforms.n = 5670) of protein coding genes (n = 3768).As we were interested in analyzing functionally relevant isoforms, we filtered for robustly expressed isoforms by imposing TPM and usage cutoffs. For the 3ʹUTR ApA isoforms, an isoform that was expressed with at least 3 TPM and with usage of 0.1 or more became part of the atlas. To focus on the most confident IpA isoforms, an IpA isoform was considered to be robustly expressed only when it was expressed with 5 TPM or more and had 0.1 usage in at least one sample. The interquartile range of the start position of the reads was also required to be 5 or more for the peaks in that particular sample to be defined as a real IpA isoform to eliminate peaks originating from PCR duplicates. These criteria helped to filter the lowly expressed isoforms as well as any possible known artifacts. Filtering for these expression criteria shrunk the atlas from 410,404 peaks to 46,923. As we were interested in IpA and 3ʹUTR ApA isoforms that would have different functional consequences, peaks that were within 200 nt were clustered to represent a single 3ʹ cleavage event. Clustering reduced the number of peaks to 40,105. After following the steps above, the atlas comprised 27,927 peaks in 15,670 genes for cleavage events of the 3ʹUTRs and 3ʹ ends of 5957 IpA isoforms in 3945 genes. For downstream analysis, we only focused on the IpA isoforms (n = 5670) with external sources of evidence as described below.We tried to corroborate the robustly expressed IpA events described thus far (n = 2241) of all the IpA events fell in the vicinity of annotated 3ʹ end (using the previous definition) based on an external annotation.1. External annotation: As annotated, we consider mRNA isoforms present in RefSeq, UCSC, or Ensembl. Last exons of all the existing transcripts of the hg19 annotation for Refseq, UCSC, and Ensembl were obtained. These last exons were resized to include a region 100 nt downstream of the annotated end. If the 3ʹ end of the IpA isoform detected by our 3ʹ-seq analysis overlapped with an expanded last exon, then it was considered to be substantiated by an external annotation. Thus, 39.52% (17 was then tested (FDR-adjusted P < 0.1). Not all IpA isoforms could be tested by DESeq. IpA isoforms where the defined windows overlapped with an annotated exon were excluded from further analysis. In total, 4802 events were tested. As a control for this analysis, random introns of expressed genes that did not contain 3ʹ end peaks were sampled and analyzed as described above. DEseq analysis returned P values consistent with the null hypothesis  could be validated by this approach.2. RNA-seq GLM: RNA-seq read coverage is expected only over the exons and not over the introns, since the splicing machinery splices out introns during co-transcriptional processing of the pre-mRNA. However, if there is an IpA isoform that ends in an intron, then there should be RNA-seq read coverage before the 3ʹ end of the IpA isoform and no read coverage after the 3ʹ end Fig. . To test18, we include the IpA event in our atlas of high-confidence IpA events. This led to the inclusion of 1332 IpA isoforms. The peaks reported by Gruber et al.18 were resized to be 75 nt width . Overall, 70% (n = 3999) of IpA events were supported by other 3ʹ-end sequencing protocols.3. Other 3ʹ end sequencing protocols: If IpA isoforms detected by our 3ʹ-seq protocol were also found by other 3ʹ-end sequencing methods65, then the IpA isoform was considered to be corroborated by polyadenylation RNA-seq reads.4. Untemplated adenosines from RNA-seq reads : In RNA-seq data, some reads may overlap the 3ʹ end of the templated transcript and the start of the polyA tail; these reads contain untemplated adenosines and thus fail to map to the genome. Reads that did not map to the human genome were therefore used to get additional support for the IpA 3ʹ ends. To make sure that these reads were at the 3ʹ end, the reads ending with 4 or more As were trimmed. Only reads that were greater than 21 nt in length after trimming were retained. Unmapped reads from all RNA-seq samples were trimmed, and all reads with untemplated As were pooled. These reads were then aligned to the human genome. Using the aligned BAM file, all the reads that were possible PCR duplicates were further filtered out. The uniquely mappable reads that overlapped with the IpA peak (20 nt extended upstream and downstream) were counted. If an IpA isoform was supported by four or more trimmed RNA-seq polyA reads together with the presence of one of the polyadenylation signals (AAUAAA and its variants)65. This step enabled us to include 323 IpA events in the atlas of highly confident IpA events.5. Highly expressed IpA isoforms: Since many of our cell types have not been previously assayed by other 3ʹ-end sequencing methods and are also not represented well in existing RNA-seq data sets, we rescued highly expressed cell type-specific IpA isoforms by using a stringent expression cutoff (10 TPM and 0.1 usage). We also required the presence of an upstream polyadenylation signal (AAUAAA and its variants)A gene is considered to be expressed if either the IpA isoform (≥ 5 TPM) or the full-length isoform (≥ 5.5 TPM) were expressed in 75% of the samples of the particular cell type.21 for 200 nt upstream and downstream of the 3ʹ ends of IpA isoforms to compare the mean conservation score of the 3ʹ ends of IpA isoforms against random introns containing polyadenylation signals, but without IpA site usage. The random introns (n = 5000) were chosen from IpA genes but we selected introns without IpA events, but with at least one polyadenylation signal (AAUAAA). One of these polyadenylation signals was randomly selected, and we obtained the phastCons 46-way conservation score for 200 nt upstream and downstream of this polyadenylation signal.We obtained phastCons 46-way conservation scores25. If a gene has multiple IpA isoforms, then the relative expression of each IpA isoform as well as the pooled full-length mRNA expression were tested independently, since different IpA isoforms are translated into different protein isoforms.IpA site usage was calculated as the fraction of reads that map to the IpA site compared to all the reads that map to the 3ʹUTR of each gene. This translates into the relative expression of the truncated protein compared with the full-length protein. To identify the statistically significant changes in the usage of pA signals, we used a GLM, where we model the read counts of all isoforms across conditions by negative binomial distributions and we test for the significance of an interaction term between isoform and condition. This form of modeling approach was adapted from DEXSeq, which is formulated for testing the differential usage of exons26. Functional annotation enrichment by DAVID was also performed for the genes that loose TMDs and retain TMDs with all the genes that have TMDs expressing IpA isoforms as background.Functional annotation enrichment was performed on the genes with significant differential usage of IpA sites Fig.  using DAhttp://www.uniprot.org/ for Homo sapiens.The information about protein domains was obtained from the UCSC UniProt annotation table (spAnnot) via the Bioconductor package-rtracklayer. Only the domains with annotation type ‘active site’, ‘domain’, ‘transmembrane region’, ‘repeat’, ‘zinc finger region’, ‘compositionally biased region’, ‘DNA-binding region’, ‘region of interest’, ‘lipid moiety-binding region’, ‘short sequence motif’, ‘calcium-binding region’, ‘nucleotide phosphate-binding region’, ‘metal ion-binding site’, and ‘topological domain’ from UniProt were used for analysis. These domains were further categorized into more broad categories: (i) Active site—active site and catalytic sites; (ii) DNA-binding domains—C2H2-type, PHD-type, C3H1-type, KRAB, Bromo, Chromo, DNA-binding, C4-type, CHCR, A.T hook, bZIP, bHLH, CCHC-type, CHCH, Bromodomain-like, CH1, C6-type, A.T hook-like, C4H2-type, and CHHC-type; and (iii) Protein–protein interaction domains (PPI)—WD, ANK, TPR, LRR, HEAT, Sushi, EF-hand, ARM, PDZ, PH, SH3, RING-type, LIM zinc-binding, WW, SH2, BTB, FERM, CH, Rod, Coil 1A, MH2, WD40-like repeat, t-SNARE coiled-coil homology, Coil 1B, Cbl-PTB, Coil, CARD, SH2-like, DED, IRS-type PTB, SP-RING-type, EF-hand-like, RING-CH-type, v-SNARE coiled-coil homology, Arm domain, LIM protein-binding, GYF, PDZ domain-binding, and PDZD11-binding. Also, if a region in protein was annotated with ‘Interaction with’ then that region was considered a PPI domain, (iv) RNA-binding domains—RRM, SAM, KH, DRBM, RBD, Piwi, PAZ, S1 motif, Pumilio, and THUMP; (v) Transmembrane domains (TMDs)—transmembrane region, ABC transmembrane type-1, ABC transporter and ABC transmembrane type-2; and (vi) Repeated—any domains that were repeated in the protein were considered repeated domains. If a gene had multiple protein isoforms, then the longest isoform was used in the analysis. The protein lengths were obtained from IpA isoforms for which there was positional information about the start of first TMD and those that retained at least one domain were used for this analysis. Further, we focused on IpA isoforms that completely lost all their TMDs due to the cleavage event in the intron. The distance of the retained CDR (in amino acids) by IpA from the first TMD was determined as: (upstream CDR from IpA−upstream CDR from the intron before the first TMD)/3.66. De novo assembly was performed on every RNA-seq sample with default settings using the hg19 RefSeq annotation. (ii) Transcripts from multiple assemblies were subsequently unified using CuffCompare, which removes redundant transcripts and provides a set of unique transcript structures67. (iii) For each individual gene, we obtained the transcripts that overlapped the gene’s coordinates. We gave preference to multi-exon transcripts over single exon transcripts. For single exon transcripts, we allowed the start/end to be within 100 nt of the transcription start site (TSS). We gave this advantage to the single exon transcripts because the direction of transcription for these transcripts is not certain. (iv) Finally, using the 3ʹ ends of IpA isoforms (from our 3ʹ-seq data), we assigned transcripts with the nearest ends to these IpA isoforms.The complete transcript structure was obtained through the following steps. (i) We used StringTie, an improved method for more accurate de novo assembly of transcripts from RNA-seq datan = 954 IpA isoforms (both annotated and unannotated). If the transcripts ends differed from the IpA 3ʹ-seq events, then we defined the 3ʹ end determined from 3ʹ-seq to be the real end. This was done as 3ʹ-seq identifies 3ʹ ends of polyadenylated mRNAs at single nucleotide resolution and thus is more accurate than transcript ends obtained from short read assembly.Firstly, we identified transcripts that ended within 50 nt of 3ʹ-seq events. If there were several assembled transcripts meeting this criterion, we chose the transcript that had the maximum number of exons. If there was a tie in the number of exons, then we chose the transcript that started closest to annotated TSS. For the remaining 3ʹ-seq events, we assigned the nearest ending transcript. Finally, using the above defined criteria for selecting transcript structures, we determined which IpA isoforms corresponded to these assembled transcripts. If the 3ʹ end of the IpA isoform was within 500 nt of the defined transcript end, then we assumed that this particular transcript represented the full structure of the IpA isoform. For some IpA isoforms we observed usage of different polyadenylation signals within the same intron. Thus, to account for such cases, for the IpA events that did not fall within 500 nt of a transcript end, we determined if it overlapped a transcript that ended within 5000 nt. If this was the case, then we assigned this transcript to that 3ʹ end. We were able to define the transcript architecture for 27. For our analysis, we considered non-coding IpA isoforms to be the ones that had coding potential probability less than 0.3, had retained coding sequence less than 25%, and had ORF ≤ 300 nt (n = 469).To determine the probability that the 5’IpA events represented non-coding transcripts, we made use of CPAT, a tool that predicts the coding potential of the transcript based on four sequence features: ORF size, ORF coverage, Fickett TESTCODE statistic, and hexamer usage bias68. As the majority of CLIP studies were performed in HEK293 cells, we focused on non-coding IpA isoforms expressed in HEK293 and only included IpA isoforms in the analysis whose exonized intron was larger than 50 nt .We used available CLIP-sequencing data of RNA-binding proteins from doRiNAZ-score of each CLIP experiment and each background region. We observed enrichment of binding sites of RNA-binding proteins in the exonized introns compared with introns and coding exons but no enrichment compared to 3ʹUTRs. The RNA-binding proteins with Z-scores ≥10 compared to introns or coding exons are PUM2, FUS, ELAVL1, TIAL1, and TAF15.We determined if the exonized part of the intron was enriched for binding sites of RNA-binding proteins compared to other regions  of the transcription units, called ‘background’ here. We calculated the expected number of binding sites in the exonized introns using each background and compared it to the observed number of binding sites in the exonized introns. This enabled us to calculate a binomial CUL4A-IpA-F1: TCGTCCCGTTTGTGTCTTCCCUL4A- IpA-R1: CCACCTGGACTCCTACGTTCCUL4A-full-length-F1: TGGAGAGAGACAAAGACAATCCGCUL4A- full-length-R1: TCATGAAGGGGAACCGTCTGIQGAP1-IpA-F1: GAGACGTCAGAACGTGGCTTAIQGAP1-IpA-R1: AATCTTCTCTCCAGCCAGCCIQGAP1-full-length-F1: GACCTAGCCAACAACAGCACIQGAP1-full-length-R1: ACAAATGTCCCATCAGAGCGAIKZF1-IpA-F1: TGGGGCTGATGACTTTAGGGAIKZF1-IpA-R1: AGTAGCCGCTTGTGTGAGAAIKZF1-full-length-F1: TTCCGTGATCCTTTTGAGTGCIKZF1- full-length-R1: CTCGCGTTATGTGCGACGAcDNA was synthesized from 200 to 1000 ng of total RNA using a SuperScript VILO cDNA Synthesis Kit  and random primers. qRT-PCR was performed using SYBR Green PCR Master Mix  on an Applied Biosystems PRISM 7900HT real-time RT-PCR machine using the following primers:−ΔΔCTIpA/(2−ΔΔCTIpA + 2−ΔΔCTfull-length) for each sample.Data are represented as the ratio of 2n bps (at least 50 bp) without running into an upstream exon from the end 3ʹ-seq peak. Similarly, full-length expression was defined as length normalized reads counts within 500 bp region from the start of the last coding exon of the gene. IpA isoforms that had significantly differential usage (FDR-adjusted P < 0.05) in MM when compared to PCs with the difference in usage >0.25 were used (n = 114). Only the IpA isoforms that had Pearson's correlation r > 0.75 (n = 28) were used to define the gene signature. Using this gene signature the patient cohort was defined segregated in two groups with low and high IpA usage, 139 and 176 respectively. Progression-free survival data were available for a subset of these patients in group A (n = 126) and group B (n = 160). Heterogeneous samples from the two groups were removed based on the group of neighboring samples. For every sample, the nearest five samples were determined based on the Euclidean distance and the sample for which the nearest 80% samples had the same group were retained, group A-filtered (n = 73) and group B-filtered (n = 115). Progression-free survival data were available for a subset of these patients in group A-filtered (n = 64) and group B-filtered (n = 100). The R survival package was used to perform the Kaplan–Meier analysis for the two groups.An independent cohort of 319 RNA-seq of MM patients with progression-free survival data for 286 patients was used for this analysis. IpA usage of the isoforms was calculated using RNA-seq data. Expression of IpA isoforms was defined as length normalized reads counts that mapped within 500 bp upstream or 52. To avoid genes with a complex genomic architecture, we removed genes that overlap with other genes in either the sense of antisense strand. An intron was categorized as retained if it satisfied the following criteria. (i) There should be at least three reads spanning both the upstream and downstream exon–intron junction. (ii) At least 50% of the intron length should be covered by 3 or more unique reads. Mappability of introns could be a limitation in this case, and thus we focused only on introns that had at least 50% uniquely mappable sequence relative to its complete length. (iii) To ensure adequate expression of the flanking exons, the median coverage over the flanking exons was required to be 10 reads or more. (iv) Since the introns should have more coverage than background noise, we considered introns to be retained if the ratio of median coverage over the intron to median coverage of the upstream and downstream exons was at least 10%.Retained introns were identified using a modified version of the IRFinder algorithm65.An intron was annotated as retained if it fulfilled all criteria in at least 66% of the RNA-seq samples of a given cell type. Introns retained in 33% or fewer samples were flagged as not retained while the introns that were retained in more than 33% samples but less than 66% of RNA-seq samples were removed from the analysis. For a 3ʹ end of an IpA isoform to occur in a particular intron, the intron must contain a polyadenylation signal or one of its variantsOur data showed that some genes had very high coverage over almost all the introns of the gene, presumably due to sequencing artifacts. We determined the /, and if this ratio was ≥0.2 then these genes were flagged for removal.The number of introns that would have IpA and IR simultaneously by chance were calculated as: probability of IpA × probability of IR × number of expressed introns with polyadenylation signal.GSE111310.All 3ʹ-seq and RNA-seq data generated for this study have been deposited in the Gene Expression Omnibus database under accession number Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data1


Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcRACME and the arcACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcRACME protein binds to the promoter region of the arcACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes. Staphylococcus aureus USA300 clone is characterised by its resistance to most β-lactam antibiotics, virulence, global spread, and association with invasive diseases (S. aureus has a constitutive arginine deaminase (ADI) pathway encoded by the arginine catabolism operon (arccons)  and has also broadly spread into prokaryotes, mainly facultative anaerobes such as Streptococcus spp., Bacillus spp., and Staphylococcus spp. among others (diseases . Under srccons) . This pag others .arc operon, which is transported in the arginine catabolism mobile element (ACME) (arc operon in ACME (arcACME) and constitutive arc operon (arccons) have the same structural genes (arcABDC), they have different genetic arrangements  of 690 bp inserted into its central region coding for a putative ArcR protein, as opposed to arcR in the constitutive operon; that is located downstream and has an independent promoter region  . Althougngements . These gngements . In addir region  Fig. 1)arc operoarccons . The bioarcR in transcription of the arcACME operon, total RNA was extracted using the TRIzol method from anaerobic trypticase soy broth (TSB) cultures  supplemented (or not) with arginine  and incubated at 37ºC for 20 h. Prior to all the experiments, RNA was treated with DNase (Promega), and the presence of genomic DNA contamination was ruled out because no gyrB gene amplification was detected by polymerase chain reaction (PCR) with the RNA as template. Integrity of the (DNA-free) RNA was assessed by agarose gel electrophoresis. From these RNA samples, cDNAs were synthesised using reverse transcriptase , a 1:4 ratio of random hexamers to RNA, 0.5 mM dNTPs, and 1X buffer  in a final volume of 25 μL [adjusted with diethyl pyrocarbonate (DEPC)-treated water]. This reaction was allowed to proceed for 1 h at 37ºC. Quantification of relative expression of constitutive arcR and arcC genes (arcCcons and arcRcons) and the genes from ACME  was determined by quantitative PCR (qPCR) to treated (50 mM arginine) and untreated (without arginine) S. aureus USA300_FPR3757 and NCTC8325 strains  strains in TSB cultures  was produced and purified from Escherichia coli BL21 (DE3) cells following the manufacturer recommendations (Thermo Scientific) . Protein expression was confirmed by western blotting using the monoclonal anti-6x-His tag antibody . The possible binding of rArcRACME to this biotinylated DNA was evaluated by means of electrophoretic mobility shift assay (EMSA), and formation of the protein-probe complex was detected by chemiluminescence. Specificity of the binding was assessed using specific and non-specific unbiotinylated competitors. In addition, the biotinylated probe and recombinant protein were cross-linked by mixing them followed by irradiation with UV light for 10 min. This product was separated in duplicate by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) and nylon membranes for western blotting and chemiluminescence assays, respectively.In order to assess the role of cultures . Relatives , showed 40% sequence similarity with ArcRcons from the constitutive operon (GenBank accession number: WP_000138214.1) in S. aureus and 99.9% identity with the orthologous protein identified in the S. epidermidis ATCC 12228 (GenBank accession number: NP_763659.1). Additionally, bioinformatics analysis of the ArcRACME protein identified a cAMP-binding domain from the CRP family (Conserved Domains Database accession number: COG0664) and a C-terminal helix-turn-helix DNA-binding motif (Conserved Domains Database accession number: cl21459), which are reported as defining characteristics of CRP/FNR transcription factors . However, transcription basic elements and a putative binding site for CRP-regulatory proteins (TTGTGA-N6-TCACA) were found to be conserved . These results confirmed the specific binding of rArcRACME to the promoter region of the arcACME operon. Furthermore, rArcRACME incubated with the promoter region probe  through the self-activation related to a positive feedback model. This mechanism can be a contributing factor of the successful adaptation of pandemic clone USA300.Currently, the influence of ADI on the pathogenicity of the USA300 clone (in the context of colonisation and infection) is unclear. Nevertheless, several studies suggest that the pathogenicity of this clone is mediated by its increased virulence (up-regulation of some virulence genes), which is best viewed as an adaptation to the hostile environment of the host and host’s antibacterial defences. For this reason, changes in concentration of oxygen in the medium can control virulence factor expression and the capacity for colonisation in hostile environments (