Long interspersed nuclear elements  have been recently implicated in the regulation of mammalian transcriptomes.F and TF) contain, in addition to those on their sense strands, conserved functional splice sites on their antisense strands, which trigger multiple exonization events. The latter is particularly intriguing in the light of the strong antisense orientation bias of intronic L1s, implying that the toleration of antisense insertions results in an increased potential for exonization.Here, we show that members of the three active mouse L1 subfamilies  has significant potential to shape the mouse transcriptome by continuously generating insertions into transcriptional units. LINE-1 elements (L1s) are by far the most abundant class of active autonomous transposons in mammalian genomes ,3 outnumThe process by which L1 sequences inserted within introns are recruited into a mRNA, termed exonization, has been primarily studied by analysis of the human transcriptome -16 but tUsing L1Xplorer , a suiteF and TF monomers (F-type) as well as unrelated A-type monomers. Consequently, mouse L1s are classified into subfamilies based on the type of the monomer they harbor [F subfamily agrees with earlier estimates [F and A subfamilies is ~1.6-fold higher than previously , followed by the A (~4.4 monomers) and TF (~4.1 monomers) families. The annotation of promoter regions is available at [Since L1 activity is expected to correlate with L1 expression level and the latter has been shown to correlate with the length of the internal promoter , we aimelable at .F, GF and A) contain functional splice sites. We mined cDNA databases (RIKEN and NCBI) to discover putative mouse exonization events involving these sequences . An overview of the different L1 exonization scenarios identified in this study is presented in Fig. Clearly any dispersed repeat commonly found in intragenic regions has the potential to be exonized due to the fortuitous occurrence of splicing signals in its sequence . BecauseFig. Antisense splice sites vs. antisense L1 insertional bias and Conclusion).Of the 52 discovered events 43 involved the exonization of antisense L1 sequences. Similarly, the authors of a very recent study investigated exonization of transposable elements and reported the greater number of the antisense orientation L1 exonization events . The obsThe most frequently used acceptor and donor splice sites we have identified are SA-154 (located in the antisense strand of 5' UTR) and SD+52 (in the antisense strand of ORF1), supported by 13 and 16 different cDNA transcripts, respectively Fig. .F element within an intron of the beige gene caused a disease-specific mutation in mouse [A classic example of sense orientation L1 exonization was previously reported when the insertion of a ~1100 bp 3' fragment of a L1 Tin mouse . Usage oin mouse ).Clearly, truncated and rearranged L1s provide different splice sites. The shortest exonized L1 insertion we have annotated is only 164 bp long (see online annotation of AK032656) and provides the antisense SA+5614 site located at the polyadenylation signal . As shown in this study (see ) the divF insertion is used to link L1-derived sequence with downstream exons encoding the C-terminus of DNA directed polymerase iota (gi: 6755273). This mRNA transcript, would encode a 254 amino acid (a.a.) protein containing two ubiquitin-binding motifs (UBM) [F. insertion, SA+120 and SD+52 generate an L1-derived exon. A similar exonization pattern, leading to first exon generation, is also evident in the transcript AK015267 where another ~6 kbp L1GF insertion provides SD+288, SA+106 and SD+52 sites, as well as SA-154. In the latter case SA+120 is substituted for the SA+106 site located nearby (see online annotation).As shown in Fig. Provocatively it has been proposed that, in general, repeat exonization via alternative splicing may constitute a vehicle for the exaption of repeat sequences into novel functions ,26,27. IF monomers. The antisense sequences of each of the three monomers can be translated into peptides which are ~60 a.a. in length yielding a novel 174 a.a. long C-terminus of GBP-5 protein  contain an L1-derived exon which contains sequences from three GTo examine the exonization potential of active L1 elements, we analyzed the residues at the positions corresponding to the cDNA-identified splice sites in a set of 2382 sequences of potentially active L1s. For this, we utilized the L1Xplorer annotation pipeline and created a set of customized modules . Since all of the identified splice sites were classical GT-AG sites, we defined the presence of these nucleotides as a criterion for a functional splice site. In a total of 2382 full-length intact LINE-1 elements 45471 donor and 47848 acceptor splice sites were annotated . This sequence is comprised mostly of truncated L1 sequences: according to RepeatMasker annotation of NCBIm35 (UCSC mm7) more than 92% of intronic L1 sequences are less than 1000 nt long. As shown above, even these short sequences can be subject to exonization. It is expected, however, that intronic full-length (FL) L1 insertions have a much higher exonization potential since they contain multiple splice sites and, for example, only FL L1s will include 5' promoter regions containing splice sites.F and TF families but some (17% in sense and 16% in antisense) belong to the inactivated F subfamily  L1 insertions. As revealed by our L1Xplorer annotation, these belong in large part (75% in sense and 78% in antisense) to the active A, GBy analysing three groups of gene annotations (cDNA and corresponding DNA), we identified as many as 1259 Ensembl Genes, 1436 UCSC Known Genes and 858 RefSeq Genes with at least one FL antisense intronic L1 insertion and 718 Ensembl Genes, 797 UCSC Known Genes and 464 RefSeq Genes that contained at least one intronic FL sense L1 insertion. Hence, the prediction of potential splice sites within FL intronic L1s may be an important consideration for researchers studying transcripts of particular genes. We have added the annotation of intronic FL L1 elements to L1Base, which is available at [The existence of antisense splice sites is highly intriguing, particularly in the light of ~2 fold antisense orientation bias of L1s located in introns of transcriptional units . This orHowever, this global picture, which is based on all L1 insertions, does not provide information on whether the bias results from processes acting on a long time scales or rather is already reflected in young L1 insertions. To gain insight into this issue we specifically looked at young intronic FL L1 insertions.2: p < 0.0001), where either orientation is equally likely.The ratio of antisense to sense FL intronic insertions is ~1.7 and chi-square testing established that this ratio is significantly different from random insertion orientation model (chicis-preference towards their encoding RNA [2: p < 0.0001). This result suggests that if insertion orientation is random for de novo insertions, and the observed bias towards antisense insertion occurs due to selection against sense insertions, this selection process is rapid.We set out to investigate, if this insertional bias is still evident among younger intronic insertions. For this analysis we utilized the set of the potentially active L1s (i.e. full-length elements with intact ORFs) that were inserted within introns. This is more stringent than analysing FL insertions with disrupted ORFs, since elements with intact ORFs are likely to be younger due to the L1 proteins' ding RNA . ~28% . One might argue here that negative selection appears to have acted specifically on the intronic sense L1 insertions belonging to the A subfamily, but it is not clear why this might be.The family distribution for the antisense intronic L1 insertions  and sense (1014) intronic full length L1s (A) and full length intergenic L1s (10671) (B) among subfamilies. See online annotation at .Click here for fileA. L1 insertions found at +/-50 kbp from TSS and TES of transcriptional units. B. Intronic-only L1 insertions. X-axis of the left-hand chart in A and B: distance from TSS, X-axis of the right-hand chart in A and B: the distance from TES. The primary (leftmost) Y-axis shows the number of nucleotides (base pairs) of L1 sequence in sense (red) and antisense (yellow) orientation. The secondary (rightmost) Y-axis: shows the ratio of antisense to sense insertions (black line). Data are plotted in bins of 100 bp. Web data: Annotation of 52 exemplary cDNAs is available at [Distribution of Line-1 elements around the transcriptional start sites (TSS) and transcriptional end sites (TES) of ~80000 transcriptional units . lable at . The datlable at .Click here for file


Childhood sarcoidosis is a rare multisystemic granulomatous disorder of unknown etiology. In the pediatric series reported from the southeastern United States, sarcoidosis had a higher incidence among African Americans. Most reported childhood cases have occurred in patients aged 13–15 years. Macrophages bearing an increased expression of major histocompatibility class (MHC) II molecules most likely initiate the inflammatory response of sarcoidosis by presenting an unidentified antigen to CD4+ Th (helper-inducer) lymphocytes. A persistent, poorly degradable antigen driven cell-mediated immune response leads to a cytokine cascade, to granuloma formation, and eventually to fibrosis. Frequently observed immunologic features include depression of cutaneous delayed-type hypersensitivity and a heightened helper T cell type 1 (Th1) immune response at sites of disease. Circulating immune complexes, along with signs of B cell hyperactivity, may also be found. The clinical presentation can vary greatly depending upon the organs involved and age of the patient. Two distinct forms of sarcoidosis exist in children. Older children usually present with a multisystem disease similar to the adult manifestations, with frequent hilar lymphadenopathy and pulmonary infiltrations. Early-onset sarcoidosis is a unique form of the disease characterized by the triad of rash, uveitis, and arthritis in children presenting before four years of age. The diagnosis of sarcoidosis is confirmed by demonstrating a typical noncaseating granuloma on a biopsy specimen. Other granulmatous diseases should be reasonably excluded. The current therapy of choice for sarcoidosis in children with multisystem involvement is oral corticosteroids. Methotrexate given orally in low doses has been effective, safe and steroid sparing in some patients. Alternative immunosuppressive agents, such as azathioprine, cyclophosphamide, chlorambucil, and cyclosporine, have been tried in adult cases of sarcoidosis with questionable efficacy. The high toxicity profile of these agents, including an increased risk of lymphoproliferative disorders and carcinomas, has limited their use to patients with severe disease refractory to other agents. Successful steroid sparing treatment with mycophenolate mofetil was described in an adolescent with renal-limited sarcoidosis complicated by renal failure. Novel treatment strategies for sarcoidosis have been developed including the use of TNF-alpha inhibitors, such as infliximab. The long-term course and prognosis is not well established in childhood sarcoidosis, but it appears to be poorer in early-onset disease. Sarcoidosis is a multisystem systemic granulomatous disease of unknown etiology that most commonly affects young adults, who frequently present with hilar lymphadenopathy, pulmonary infiltration, and ocular and cutaneous lesions . AlthougDespite a variety of hypotheses regarding causative agents, the cause of sarcoidosis is unknown . The defIn this review, we discuss the epidemiology, pathogenesis, etiology, and clinical features of sarcoidosis in children, and examine the current approaches to diagnosis and treatment of this enigmatic disease.The prevalence of sarcoidosis in the adult population ranges from 10 to 40 per 100,000 in the United States and Europe [Most reported childhood cases have occurred in patients aged 13–15 years ,12. In aAdult studies have reported a slightly higher disease rate for women. A population-based study of incidence and survival in adults with sarcoidosis reported incidence rates of 5.9 per 100,000 person-years for men and 6.3 per 100,000 person-years for women. No clear sex predominance exists in childhood sarcoidosis. In a recent study from Denmark, the male/female gender ratio was close to one .The racial distribution of sarcoidosis varies with geographic location. In the US adult population, sarcoidosis occurs in about 35.5 per 100,000 blacks and 11 per 100,000 whites. Studies in military and veteran populations showed that blacks are 10–17 times more commonly affected with sarcoidosis than are whites . In chilWithin the US, approximately 80% of childhood cases have been reported primarily in Virginia, North Carolina, South Carolina, and Arkansas, and Louisiana, suggesting that the southeastern and south central states are an endemic area for childhood sarcoidosis ,14,16,17Familial aggregation and the striking racial variation in sarcoidosis incidence suggest a genetic predisposition to develop sarcoidosis . The strGenetic factors may also be important in defining the pattern of disease presentation, its severity, and prognosis. Several investigators have searched for associations with human leukocyte antigen (HLA)-related genes that may confer susceptibility to sarcoidosis . A genomDespite the global occurrence of sarcoidosis, the etiology of sarcoidosis is unknown. Most experts think that sarcoidosis results from exposure of genetically susceptible hosts to specific environmental, occupational or infectious agents that trigger an exaggerated cellular immune response, leading to granuloma formation ,26. DespMycobacterium tuberculosis and other atypical species have received the greatest amount of attention [Case clustering, and immunologic and clinical similarities of sarcoidosis to infectious granulomatous diseases suggests that an infectious agent may be responsible. Mycobacteria, including ttention -32. The ttention .Mycobacteria, many different antigens are under suspicion, including other bacteria such as Proprionibacterium acnes [Besides um acnes . Howeverum acnes . Recent um acnes .+ Th (helper-inducer) lymphocytes. This results in proliferation and activation of the T cell [Sarcoidosis is a chronic inflammatory disease characterized by a highly focused exaggerated immune response to an unknown antigen(s) at the target organs. The hallmarks of the disease, sarcoid granulomas, most likely are formed in response to a persistent, poorly degradable, antigenic stimulus . Macrophe T cell .+ and CD8+ T-lymphocytes, as well as a few B-lymphocytes, form a characteristic ring at granuloma periphery [The immunopathology has been best studied in bronchoalveolar lavage fluid and lung disease in which early lesions consist of an alveolitis with a high proportion of activated CD4+ Th1 cells, which may precede granuloma formation . CD4+ aneriphery . Most greriphery . Additioeriphery ,37. IL-1eriphery .Chemokines may play an important role in the pathogenesis of sarcoidosis . ChemokiThe CD4+ lymphocytes, in association with other immune effector cells, such as macrophages, mast cells, and natural killer cells, perpetuate the inflammatory response by release of cytokines, monocyte chemotactic factors, macrophage migration inhibitory factor, leukocyte inhibitory factor, adhesion molecules , and growth factors ,36.As a result of these various immunologic interactions, an acute and often a chronic cascade of inflammation occur. This is characterized by changes in tissue permeability, cellular influx, and local cell proliferation, resulting in a granuloma. Persistent antigenic stimulation is believed to maintain the pathogenic processes. Sarcoid granulomas either resolve or heal by fibrosis. Genetic polymorphisms may influence the clinical expression of sarcoid granuloma and disease outcome .Other immunologic abnormalities observed in patients with sarcoidosis include circulating immune complexes, B-cell hyperactivity, spontaneous in situ production of immunoglobulins, and depression of cutaneous delayed-type hypersensitivity reactions .Because sarcoidosis is a multisystem disease and affects most organs, the clinical presentation can vary greatly. In most children, the disease frequently involves the lungs, lymph nodes, eyes, skin, liver, and spleen ,7,9. MosTwo distinct forms of childhood sarcoidosis appear to exist. Older children usually present with a multisystem disease similar to the adult manifestation with frequent lymphadenopathy and pulmonary involvement, as well as generalized signs and symptoms, such as fever, malaise, and weight loss ,13-15. IThe lung is the organ most commonly involved in sarcoidosis ,2,7. PulIn a recent Danish study, chest radiographs were normal (stage 0) in 10%; 71% had isolated bilateral hilar lymphadenopathy (stage I), 8.3% had bilateral hilar adenopathy with pulmonary infiltrates (stage II) and 8.3% had parenchymal infiltrates without hilar adenopathy (stage III). None of the 48 reported patients had evidence of fibrosis (stage IV) [Nearly half of all children with sarcoidosis demonstrate restrictive lung disease on static and dynamic pulmonary function tests ,7,11,43.The most common physical sign of reticuloendothelial involvement is peripheral lymph node enlargement noted in 40% to 70% of cases ,14, whicOcular involvement is common in childhood sarcoidosis. Visual symptoms such as eye pain, blurry vision, photophobia, and redness may be present in 29% of the patients ,44. AnteAn erythematous rash is commonly noted in childhood sarcoidosis and occurs in 77% of young children and 24–40% in older children ,11,14 F. The mosMusculosketelal features of sarcoidosis include joint effusions, joint pain, and, rarely, osseous involvement. Arthritis has been reported in 15% to 58% of children with sarcoidosis ,5,11. ThRenal involvement occurs infrequently in children with sarcoidosis ,15,47. TDerangement in calcium metabolism manifesting as hypercalcemia and/or hypercalciuria, occurs in up to 30% of children, and can be associated with nephrocalcinosis and nephrolithiasis ,11,48. IParotid gland enlargement is a frequent finding in children with sarcoidosis, especially the early onset type . SarcoidEarly-onset childhood sarcoidosis, i.e., with onset in the first 5 years of life differs from sarcoidosis in older children and adults and often poses a diagnostic challenge to the clinician ,10,15. TCARD15) in 50–90% of cases [In 1985, Edward Blau described families with autosomal dominant granulomatous disease demonstrating the classic triad of arthritis, dermatitis, and uveitis, an identical phenotype to early-onset sarcoidosis . Recent of cases . This fiof cases . Althougof cases  found noEarly-onset sarcoidosis may be overlooked due to its similarity to systemic-onset JRA . Both enThere is no laboratory test diagnostic of sarcoidosis. Laboratory evaluation may reveal elevated erythrocyte sedimentation rate (ESR) or other acute phase reactants. Anemia, leukopenia, and eosinophilia are commonly seen on blood counts . ImmunolThe source of serum ACE in sarcoidosis is believed to be epitheloid cells in granulomata. ACE serum levels have been shown to be useful in diagnosing sarcoidosis and following disease activity and the effect of therapy in older children with sarcoidosis ,58. RefeChest radiograph is very useful and may reveal bilateral hilar adenopathy ,42. BecaThe diagnosis of sarcoidosis is confirmed by demonstrating a typical noncaseating epithelioid cell granuloma on a biopsy specimen  Figure . To furtOccasionally, sarcoidosis may present initially as fever of unknown origin (FUO) and localized bone pains, mimicking a number of infectious, inflammatory, and neoplastic processes. In such cases, magnetic resonance imaging (MRI) may reveal multifocal small nodular lesions within the marrow and help localize lesions for diagnostic exploration .The differential diagnosis depends largely on the clinical presentation of sarcoidosis . It is cThe current therapy of choice for childhood sarcoidosis with multisystem involvement is corticosteroids ,9,15,63.Some patients may relapse, either during steroid taper or after discontinuation of the drug. In such cases, steroid treatment should be restarted with a dosage similar to that used in the induction treatment . Few patOther immunosuppressive agents, especially low-dose methotrexate (MTX) have been used to treat adult patients with sarcoidosis who have steroid-resistant disease or in those with unacceptable adverse effects from glucocorticoids with good success . In 1997Alternative immunosuppressive agents, such as azathioprine, cyclophosphamide, chlorambucil, and cyclosporine, have been tried in adult cases of sarcoidosis with questionable efficacy . The higThe prognosis and natural history of sarcoidosis in children is unclear because of the rarity of the disease and the small number of reported series. ,11,69,70In one series, Marcille et al  performeSarcoidosis in very young children with involvement of the eyes, joints and skin have a guarded prognosis with the likelihood of a chronic progressive course ,6; 80–10The authors declare that they have no competing interests.The parents signed a consent and gave permission to use the photos in the manuscript.


Dear Sir,I read with interest the article ‘Tuberculous Sarcoidosis’ by Shah JRAuthor has stressed on considering ‘tuberculous sarcoidosis’ as a definite clinical entity where two diseases present simultaneously or another disease develop in a course of existent disease. I thanks the author, for awaring the clinicians about this distinct entity for personal reason, that now I can suspect retrospective diagnosis of ‘tuberculous sarcoidosis’ in three cases in my previous clinical experience. Two AFB smear positive and tuberculin reactive pulmonary cases with noncaseating granulomas on tissue biopsy, required oral corticosteroid in view of poor response with antituberculosis drugs and another case, a 10 year old male child with lymphocyte predominant exudative pleural effusion and tuberculin reactivity developed cervical lymphadenopathy, nine months after completion of six months antituberculosis therapy with adequate response. The scalene node biopsy showed noncaseating granulomas and a second course with antituberculosis drugs resulted in poor response and patient lost to follow up. Although none of these cases could be investigated for sarcoidosis as diagnosis of tuberculosis was confirmed and the term ‘tuberculous sarcoidosis’ was neither familiar to me nor is mentioned in the standard textbooks of respiratory medicine till date. Since tuberculosis is rampant in our country and may present with various unusual clinical and radiological features, the diagnosis of other coexistent diseases is often overlooked. Lack of infrastructure, financial constraints, and lack of newer diagnostic techniques at most centre of our country, despite adequate clinical material and trained medical persons are possible causes for these situations. The recent studies on possible association of tuberculosis and sarcoidosis are interesting and encouraging further research in this field.+ CD4+ T cells at the sites of granulomatous inflammation, consistent with a MHC-restricted antigen driven processSarcoidosis has pathologicOne of the strongest arguments against a potential role of mycobacteria in sarcoidosis pathogenesis is inability to detect microorganism on histological staining or by culture from pathologic tissues. The possible explanations for negative microbial workup noted in sarcoid lesions could be very low concentration of the bacteria's, ultra-slow growth pattern, and lack of certain nutritional requirements etc. or the sarcoidosis pathogenesis may reflect an immune response to infectious antigens that might not be dependent upon actively replicating organisms. Recently newer diagnostic techniques have been emerged for assessing presence of microorganisms in pathologic tissues. Polymerase chain reaction (PCR) analysis of pathologic tissue for 16S r RNA serve as an alternative means of identifying putative infectious agents that are difficult to isolate. One study of PCR analysis for conserved Mycobacterium 16S r RNA and rpo B sequences revealed the presence of slow-growing mycobacteria such as M tuberculosis and M avium, as well as unique 16S r RNA and rpo B sequences that suggest a novel mycobacteriumAnother mechanism to identify the causative microorganism is use of antigen specific immune responses to microbial antigens and this has been. recently utilized to identify novel infectious agent in SARSThese recent studies of successful molecular analysis and humoral immunity to mycobacterial antigens from sarcoidosis patients have renewed interest in a potential role of mycobacteria in sarcoidosis and support the hypothesis that mycobacterias may have a causal role in ‘some’ sarcoidosis patients. Further molecular studies with positive and negative controls and investigation of genetic risk factors will be important, in order to explain why some patients are found to have an association with microbial antigens and others are not. In conclusion, we must be flexible on the diagnosis of two co existent diseases and possibility of ‘tuberculous sarcoidosis’ may be considered whenever such clinical situation arises and supported by complete investigation backup.


Lactococcus lactis and ii) the ability of the resulting recombinant strain to produce either capsomer-or virus-like particles (VLPs).The expression of vaccine antigens in lactic acid bacteria (LAB) is a safe and cost-effective alternative to traditional expression systems. In this study, we investigated i) the expression of Human papillomavirus type 16 (HPV-16) L1 major capsid protein in the model LAB L. lactis, respectively. The capacity of L. lactis harboring either pCYT:L1 or pSEC:L1 plasmid to accumulate L1 in the cytoplasm and supernatant samples was confirmed by Western blot assays. Electron microscopy analysis suggests that, L1 protein produced by recombinant lactococci can self-assemble into structures morphologically similar to VLPs intracellularly. The presence of conformational epitopes on the L. lactis-derived VLPs was confirmed by ELISA using an anti-HPV16 L1 capsid antigen antibody. Our results support the feasibility of using recombinant food-grade LAB, such as L. lactis, for the production of L1-based VLPs and open the possibility for the development of a new safe mucosal vector for HPV-16 prophylactic vaccination.HPV-16 L1 gene was cloned into two vectors, pCYT and pSEC, designed for controlled intra- or extracellular heterologous expression in  Therefoe.g. administered by oral, intranasal, rectal or vaginal route) are more convenient than systemic vaccines, because they are easier to administer, relatively cheap to produce and less invasive, an especially important criteria when used with children and immunosuppressed patients [Alternatively, mucosal vaccines  via heterologous LAB, including Lactobacillus casei and L. lactis, has been previously shown to generate VLPs [L. lactis is able to produce HPV-16 L1 protein using the NICE system [L. lactis is able to self-assemble into either capsomers or VLPs.Lactic acid bacteria (LAB) are non-pathogenic and non-invasive Gram-positive bacteria considered as being promising candidates for controlled and targeted administration of vaccine antigens to the mucosal immune system -9. Becauodel LAB -12. Muco in mice ,13. Thisate VLPs ,15. TherE system  and whetL. lactis to produce and secrete L1 protein was examined using lactococci strains harbouring pCYT:L1 and pSEC:L1 plasmids . In the absence of nisin, no L1 signal was detected in either L. lactis (pCYT:L1) or L. lactis (pSEC:L1) strain, indicating that NICE system allows tight control of gene expression. Induced cultures of L. lactis (pCYT:L1) strain resulted in a clear band at the expected size for native L1 (~58 kDa) was observed in the cell fraction whereas no signal was detected in the supernatant. Similar analysis of L. lactis (pSEC:L1) resulted in a clear band in the cell fraction corresponding to SPUsp45-L1 precursor . Unfortunately, since a major non-specific band (which migrates at the expected size for native L1: ~58 kDa) reacts with anti-L1 antibodies in supernatant samples, L1 secretion cannot be determined. This non-specific band most probably corresponds to Usp45, the predominant L. lactis-secreted protein, and frequently detected by immunoblotting when using protein G-horseradish peroxidase conjugate [The capacity of onjugate ,16.L. lactis (pSEC:L1) strain, we modified the protocol of immunoblotting by using a higher dilution of protein G-horseradish peroxidase conjugate  and a higher concentration of HPV-16 L1-specific monoclonal antibody (0.5 μg/ml). We also used a higher dose of nisin (ie. 10 ng/ml) for the induction of L1 expression. As shown in Figure Usp45-L1 precursor , and a weak band in the supernatant fraction corresponding to the secreted mature L1 protein. Smaller-sized L1 products are also observed, reflecting degradation by HtrA or ClpP proteases or incomplete L1 synthesis at high expression levels (ie. 10 ng/ml of nisin). L1 secretion appears to be low, as about only 5% of the protein is detected in the supernatant.In order to better determine the secretion of L1 protein by recombinant L. lactis (pCYT:L1) strain and have similar sizes (30–50 nm in diameter) and morphologies as VLPs produced in other prokaryotic and eukaryotic systems (~55 nm) [L. lactis (pSEC:L1) strain  [L. lactis (pGK-)  ,15,17-20cretion) , suggestL. lactis-produced VLPs, an ELISA assay was performed using a monoclonal antibody that recognizes conformational epitopes present in HPV-16 VLPs . Total protein cell extracts from L. lactis strains were assayed using a conformational anti-HPV16 L1 antibody. As shown in Figure L. lactis (pCYT:L1) and L. lactis (pSEC:L1) strains resuspended in PBS pH 7.0 (assembled VLPs) react with the anti-HPV16 L1 capsid antibody, indicating that conformational epitopes are displayed on L. lactis-derived VLPs. Note that the L. lactis (pCYT:L1) ELISA signal is significantly higher with L. lactis (pSEC:L1) strain. This could be explained in part by a higher quantity of intracellular-VLPs in L. lactis (pCYT:L1) strain, as observed by TEM analysis  strain was designed for secretion. Hence, it does not retain much VLPS and/or L1 protein intracellularly and most VLPs are secreted to the external medium  do not react with the conformational antibody .Samples from the negative control y Figure . As expee.g. HPV-16), development of effective mucosal vaccines is advantageous.Infections with HPV are closely related to the development of CxCa . For thiL. lactis, to produce HPV-16 L1 protein and we demonstrated by Western blot analysis that full-length L1 protein could be efficiently expressed in L. lactis. We also demonstrated for the first time that L. lactis-derived L1 protein can self-assemble into structures morphologically similar to VLPs intracellularly by TEM experiments. Most important, these VLPs display conformational epitopes as confirmed by ELISA analysis. This is the first time that HPV-16 L1 protein is produced in L. lactis using the NICE system [L. lactis is a non-colonizing bacterium, a system allowing the bacterium to be preloaded, such as with NICE system, with antigen before in vivo applications  are highly desirable.In this work, we used the model LAB, E system . This syL. lactis provide a possible means for targeting L1 protein to humans. However, the use of genetically modified microorganisms raises legitimate concerns about their survival and propagation in the environment and about the dissemination of antibiotic selection markers (such plasmids used in this study) or other genetic modification to other microorganisms. Thus, before recombinant bacteria could be applicable to humans a biologic containment of recombinant microorganisms is necessary [Note that recombinant ecessary .In conclusion, this study demonstrates the potential of the use of recombinant LAB for the production of HPV-16 VLPs and suggests the development of a new mucosal prophylactic HPV-16 vaccine.Lactococcus lactis strains were grown in M17 medium (Difco) supplemented with 0.5% glucose (GM17) at 30°C without agitation. Escherichia coli was grown in Luria-Bertani at 37°C with vigorous agitation. Plasmid constructions were first established in E. coli and then transferred to L. lactis by electrotransformation [L. lactis, chloramphenicol (10 μg/ml); for E. coli, ampicillin (100 μg/ml) and chloramphenicol (10 μg/ml). Isolation of plasmid DNA was performed by using a Mini-Scale purification system (QIAGEN S.A.). Lysozyme (10 mg/ml) was added prior to the lysis step and incubated for 30 min (37°C) to prepare the protoplasts. PCR  was performed using Vent DNA polymerase (Promega), and DNA sequences were confirmed by sequencing (MWG-Biotech AG). Restriction and DNA-modifying enzymes were used according to the supplier's recommendations.The bacterial strains and plasmids used in this work are listed in Table ormation . PlasmidNsiI-L1 HPV16 (5'-GATGCATCA CAACAGGTGACTTTTATTTACATC-3') for the coding strand and L1 HPV16 (5'-GTTACAG CTTACGTTTTTTGCGTTTAGCAG-3') for the complementary strand. The PCR product was cloned into pCR:TOPO (Invitrogen), resulting in pCR:TOPO:L1. L1 gene was then purified from NsiI/SpeI-cut pCR:TOPO:L1 and cloned into purified backbones isolated from NsiII-SpeI-cut pCYT:E7 and pSEC:E7 resulting in pCYT:L1 and pSEC:L1 , the gene encoding Usp45, the predominant L. lactis-secreted protein [Usp45 is absent. In both pCYT:L1 and pSEC:L1, expression of L1 is controlled by the PnisA promoter [L. lactis strain carrying the regulatory genes nisR and nisK (Table -).A 1,606-bp DNA fragment encoding HPV-16 L1 protein (without ATG start codon) was PCR amplified from the pcDNA3-L1 plasmid  = 0.6, followed by induction with 1 ng/ml of nisin (Sigma) for 1 h. Protein samples were prepared from 2 ml of L. lactis induced cultures. After centrifugation , the cell pellet and supernatant were treated separately. To compare the amounts of secreted and cell-associated proteins, both cell and supernatant fractions were concentrated and sample concentration calculated as follows: the equivalent of 1 ml of 1 OD600 unit of the sample (either cell or supernatant) was concentrated in a 100 μl final volume as described below, and 10 μl was loaded for SDS-PAGE. Supernatants were filtered on 0.2 μm pore-size filters  and treated with 1 mM PMSF and 10 mM DTT, followed by the addition of 100 μl of TCA (100%) to precipitate proteins. Samples were incubated for 10 min on ice, and proteins were recovered from the pellets after centrifugation at 4°C for 10 min at 13,000 rpm. The resulting pellet was dissolved in 1/20 volume of 50 mM NaOH. The cell fraction was obtained by cell lysis in 70 μl of lysis buffer  complemented with 1 mM PMSF and 10 mM DTT. After 30 min of incubation at 37°C, cells were lysed with 30 μl of 20% SDS. Equal volumes of 2× loading buffer were added to all samples and 10 μl of each sample were analyzed by SDS-polyacrylamide (10 and 12%) gel electrophoresis. Western blotting on PVDF membranes (Millipore) was carried out using an HPV-16 L1-specific monoclonal antibody . Immunodetection was performed with protein G-horseradish peroxidase conjugate (Bio-Rad) at a dilution of 1:5000 and an enhanced chemiluminescence kit (Dupont-NEN) as recommended by the suppliers.For the induction of L1 expression from the nisin promoter, strains were grown to an optical density  for 1 hour at room temperature. Samples were then post-fixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate before gradually dehydrating in ethanol (30–100%) and embedding in Epon resin. Thin sections (70 nm) were collected onto 200 mesh copper grids and counterstained with lead citrate before examination with Zeiss EM902 TEM operated at 80 kV . Images were acquired with a charge-coupled device camera (Megaview III) and analysed with ITEM Software .L. lactis-derived VLPs and L1 protein were recovered as follows: 10 ml of induced-L. lactis cultures were pelleted and resuspended in 3 ml of either PBS pH 7.0 (assembled VLPs) or in carbonate buffer  (disassembled VLPs). Cells were then lysed with 0.1 g of acid-washed glass beads . Cellular debris were eliminated by centrifugation and 200 μl of supernatant containing VLP's or L1 protein were applied on ninety-six well plates (Nunc Maxisorp) and incubated at 4°C for 8 h. Plates were washed twice with PBS plus 0.1% Tween-20 and blocked at room temperature for 1 h with 5% non-fat dry milk in PBS. After plate washing, 100 μl of either an anti-HPV16 L1 capsid antigen  or anti-L1 (BD Pharmigen) diluted 1:50 in 1% non-fat dry milk in PBS were added. After 2 h of incubation at room temperature the plates were washed and 100 μl of a secondary anti-mouse IgG antibody  diluted 1:2500 in 1% non-fat dry milk in PBS was added and the plate incubated 1 h at 4°C. The plates were washed and the reaction developed by addition of 200 μl of p-nitrophenyl phosphate substrate  dissolved at 10 mg/ml in sodium carbonate buffer. The plates were incubated at room temperature until yellow colour was developed (30–60 min). Finally the reaction was stopped by the addition of 50 μl of 2 M NaOH. The absorbance was immediately measured at 405 nm (A405 nm).ELISA analysis was performed as described previously  with somThe authors declare that they have no competing interests.NGCP and LGBH conceived and designed the study with the help of PL. LGBH constructed the plasmids and strains. NGCP performed most experiments NGCP, LGBH and PL drafted the manuscript. All authors have critiqued and approved the final manuscript.


PVs (PV) are small, non-enveloped, double-stranded DNA viruses that have been identified as the primary etiological agent for cervical cancer and their potential for malignant transformation in mucosal tissue has a large impact on public health. The PV family Papillomaviridae is organized into multiple genus based on sequential parsimony, host range, tissue tropism, and histology. We focused this analysis on the late gene products, major (L1) and minor (L2) capsid proteins from the family Papillomaviridae genus Alpha-papillomavirus. Alpha-PVs preferentially infect oral and anogenital mucosa of humans and primates with varied risk of oncogenic transformation. Development of evolutionary associations between PVs will likely provide novel information to assist in clarifying the currently elusive relationship between PV and its microenvironment  and macro environment . We attempt to identify the regions of the major capsid proteins as well as minor capsid proteins of alpha-papillomavirus that have been evolutionarily conserved, and define regions that are under constant selective pressure with respect to the entire family of viruses.This analysis shows the loops of L1 are in fact the most variable regions among the alpha-PVs. We also identify regions of L2, involved in interaction with L1, as evolutionarily conserved among the members of alpha- PVs. Finally, a predicted three-dimensional model was generated to further elucidate probable aspects of the L1 and L2 interaction. Papillomaviruses (PVs) are small, non-enveloped, double-stranded DNA viruses identified as the primary etiological agent in cervical cancer and their potential for malignant transformation in mucosal tissue is a major health concern. Papillomaviruses (PVs) have also been linked to benign cutaneous lesions and with some non-melanoma skin cancers. These viruses are very common pathogens of epithelial surfaces that account for a variety of proliferating lesions in humans and animals. In the past few years, the available number of complete HPV genomic sequences has increased substantially to comprise more than 150 GenBank entries (2007).Papillomaviridae is organized into multiple genera based on sequential parsimony, host range, tissue tropism, and histology. We focused this analysis on the late gene products, major (L1) and minor (L2) capsid proteins of the family Papillomaviridae genus Alpha-papillomavirus. Alpha-PVs preferentially infect oral and anogenital mucosa of humans and primates with varied risk of oncogenic transformation. Two members of the genus Alpha-papillomavirus are also associated with cutaneous lesions, Human papillomavirus (HPV) 2 and HPV10. The alpha genus includes twelve cutaneous HPV types and two simian PVs [The PV family , major L and minod minor L capsid pInfection with HR HPV genotypes such as HPV 16 and HPV 18 has been directly related to the subsequent development of cervical cancer ,4.Escherichia coli has recently been resolved at a resolution of 3.5-A° [PV genomes are characterized by eight well-defined open reading frames (ORFs), which are all transcribed from the same DNA strand and orientation. The translated proteins are classified as "early" (E) or "late" (L) based on their temporal expression. The viral ORFs include 3 regulatory genes involved in transcription and replication , 3 oncogenes E5, E6, and E7), and 2 genes encoding for self-assembling proteins which constitute the viral capsid (L1 and L2) , and E7,,9. The sf 3.5-A° . Moreovef 3.5-A° ,12.Conformational epitopes and the location of epitopes are critical for the production of neutralizing antibodies ,14. It hTaking these facts into consideration, we hypothesized that the interaction domains of L1 and L2 should be fairly conserved among alpha- PVs. Some L2 domains may be exposed at the virion surface, thus enabling recognition of a specific epitope for immune recognition ,21. ThisThe association of HPVs with benign and malignant neoplasia has led to research efforts focused toward improvement on the current understanding of diversity within this virus group, so that diagnosis, treatment, and control of HPV infections may be optimized. Many aspects about evolution of PVs are still relatively poorly understood. Therefore, probing of evolutionary and structural relationships between PVs will likely provide novel insight to assist in clarifying the functional differences between PVs and their tropic microenvironment; cutaneous cells or mucosal epithelial cells. To date, a broad range of bioinformatics tools have been applied to analyze the complete PV genome . In this paper we identify the regions of the major capsid proteins as well as minor capsid proteins of alpha-papillomavirus that have been evolutionarily conserved, and define regions that are under constant selective pressure with respect to the entire family of viruses. Here we show that the loops of L1 are, in fact, the most variable regions among the alpha-PVs. We also identify regions of L2 involved in interaction with L1 as evolutionarily conserved among the members of alpha-PVs. Finally, we generated a predicted three-dimensional model to further elucidate probable aspects of the L1 and L2 interaction.The Seventy-six alpha-PV sequences obtained for this analysis were retrieved from the NCBI protein database according to the reference list of alpha-PVs published on the Universal Virus Database . In addiThe compiled protein sequence sets were aligned using MUltiple Sequence Comparison by Log-Expectation, MUSCLE ,26. MUSCMUSCLE outputs were then loaded into the CLUSTALX user interface for graphical representation of residue conservation and analysis . Sequenc. The L2 amino acid sequence data was then submitted to SAM-T09, Sequence Alignment and Modeling System, for tertiary structure prediction [The HPV16 L1 protein structure was obtained from the RCSB Protein Data Bank . The secediction -34. The ediction . HPV16 Lediction -40. The We found the external loop regions of alpha-PVs correlate to the least conserved regions in our alignment Figure . The extHPV 16 cysteine 201 and 454 are conserved across the entire alpha-papillomavirus family alignment  is in aUpon analysis of the 3d docking prediction between L1 and L2, we targeted regions of L1 that were in prime positions to be involved in the protein interaction with L2, specifically the interaction in the region of 247–269 and the region of 113 to 130  that are thought to bind to heparan sulfate side chains on the cell surface and facilitate cellular entry. Mutations of these residues to alanine is known to cause a reduction cell binding and infection of pseudovirions . ResidueAlso there is a region, the H4 helix that is thought to be involved in pentamer-pentamer interaction . H4 is tWe have shown that the conserved region where the final 11 AA of the C-terminus are involved in DNA interaction  is fairlWe found the carboxy terminal L1 binding domain of L2 to be conserved among the alpha-PVs irrespective of high or low risk group. However, the structural interaction of L1 and L2 and formation of capsid is still not clear. Minor capsid protein (L2) binds the L1 capsomers but not to the VLP, suggesting that L2 co-assembles with L1 rather than being inserted into a pre-formed capsid . L2 is rTwo distinct L1 binding domains have been described for BPV1 L2; a C-terminal L1 binding domain (BPV L2 aa384–460) that interacts with L1 capsomers in vitro, and a central region (BPV L2 aa129–246) that fails to interact with capsomeres . These aThe carboxy-terminus L1 binding domain described between residues 396–439 of HPV11 L2, is consistent with the C-terminal L1 binding domain in residues 384–460 of BPV1 L2 . Our resRecently much focus has been given toward the development of potential vaccines against HPV. Anti-L1 antibodies obtained by immunizing mice or rabbits with the L1 capsids have been shown to have primarily type specific neutralizing activity. Limited cross-neutralizing activity has been observed between closely related types such as HPV18 and 45, and HPV6 and HPV11 . MoreoveSeveral studies have attempted to identify the nature of both neutralizing epitopes of both L1 and L2 using L1/L2 VLP to better define the topology of L2. All these data suggest that HPV16 L2 residues 108–120 and 69–81 are epitopes displayed on the surface of VLPs and virions . ClearlyNonetheless, our L2:L1 structural interaction model had distinct similarities with the Polyomavirus VP2 interaction with VP1 . The PolThe authors declare that they have no competing interests.JL assembled HPV sequences and performed MUSCLE and Clustal analyses. DP did further data and literature research and helped write the manuscript. SR performed 3D protein modeling and docking analyses. TJ, WAH helped in analysis of 3D structures and conserved regions. FT helped in writing the manuscript and data analysis. PCA conceived of the study and coordinated the work, performed 3D rendering of structures and using Pymol, and edited the manuscript.Multiple sequence alignment of L1 and L2 proteins. Seventy-six alpha-HPV L1 and L2 sequences were obtained from the NCBI protein database under the Universal Virus Database, as well as six known HPV-16 L1 and L2 variant sequences provided by the ICTV. The sequences were aligned using the MUltiple Sequence Comparison by Log-Expectation (MUSCLE). MUSCLE outputs were loaded into CLUSTALX user interface for graphical representation of residue conservation and analysis. Sequence conservation is by the height of residue logos (indicated in bits), as generated by WebLogo 3. All outputs for both sequences are shown.Click here for file


Based on the quantitative RT-PCR analysis, methylcellulose treatment did not influence the transcriptional expression of both authentic and codon-modified L1 genes in KCs. Western blot showed that methylcellulose significantly increased the levels of the L1 proteins expressed from two authentic L1 genes. Conversely, methylcellulose dramatically decreased L1 protein expression in KCs transfected with two codon-modified L1 expression constructs. These data suggest that L1 protein expression is associated with KC differentiation induced by methylcellulose treatment and regulated at the post-transcriptional level.Papillomaviruses (PVs) are double-stranded DNA viruses that infect keratinocytes in differentiating epithelia and induce hyperproliferative lesions. Here, we used methylcellulose to induce cell differentiation of primary mouse keratinocytes (KCs) in  Amplificription  and to acription  and to pcription ,6. Howevcription .Nat) L1 gene plasmids (pcDNA3HPV6b Nat L1 and pcDNA3BPV1 Nat L1) and two codon modified (Mod) L1 gene plasmids  were used in the experiments as previously described [Recently, we established mouse primary KCs culture system to express PV L1 proteins by transient transfection of authentic or codon modified L1 gene expression constructs . Using tescribed .We prepared primary KCs from new-born mouse skin as previously described . The pri6 cells of the freshly isolated mouse primary KCs were respectively transfected with 2 μg of each of the four PV L1 plasmid DNAs using lipofectamine . The L1-transfected KCs were incubated in basal KC medium for 18 h, and then grown in semisolid KC-SF complete medium containing different concentrations of methylcellulose  for 48 h. The L1-transfected KCs treated with methylcellulose were harvested for analysing L1 gene expression at both transcriptional and translational levels . The specificity of RT-PCR was determined by the Rotor Gene Software and agarose gel electrophoresis  declare that they have no competing interests.XW and BL conducted experiments together. XW wrote the manuscript. KNZ designed and coordinated the research efforts and edited the manuscript. All co-authors read and approved the final manuscript.


The cell adhesion molecule L1 is crucial for mammalian nervous system development. L1 acts as a mediator of signaling events through its intracellular domain, which comprises a putative binding site for 14-3-3 proteins. These regulators of diverse cellular processes are abundant in the brain and preferentially expressed by neurons. In this study, we investigated whether L1 interacts with 14-3-3 proteins, how this interaction is mediated, and whether 14-3-3 proteins influence the function of L1.in vitro phosphorylation assay, 14-3-3ζ promoted CKII-dependent phosphorylation of the L1ICD. Given that L1 phosphorylation by CKII has been implicated in L1-triggered axonal elongation, we investigated the influence of 14-3-3ζ on L1-dependent neurite outgrowth. We found that expression of a mutated form of 14-3-3ζ, which impairs interactions of 14-3-3ζ with its binding partners, stimulated neurite elongation from cultured rat hippocampal neurons, supporting a functional connection between L1 and 14-3-3ζ.By immunoprecipitation, we demonstrated that 14-3-3 proteins are associated with L1 in mouse brain. The site of 14-3-3 interaction in the L1 intracellular domain (L1ICD), which was identified by site-directed mutagenesis and direct binding assays, is phosphorylated by casein kinase II (CKII), and CKII phosphorylation of the L1ICD enhances binding of the 14-3-3 zeta isoform (14-3-3ζ). Interestingly, in an Our results suggest that 14-3-3ζ, a novel direct binding partner of the L1ICD, promotes L1 phosphorylation by CKII in the central nervous system, and regulates neurite outgrowth, an important biological process triggered by L1. L1CAM gene 1176RSL, which interacts with the adaptor protein AP-2, is necessary for clathrin-mediated endocytosis of L1 1176 by the nonreceptor tyrosine kinase p60src prevents L1 binding to AP-2 1181, the second serine residue of the YRSLESDNEE sequence in the L1ICD, can be phosphorylated by CKII L1 is a cell adhesion molecule of the immunoglobulin superfamily which is essential for normal development of the mammalian nervous system. Constitutively L1-deficient mice display severe brain malformations, in particular hydrocephalus and agenesis of the corpus callosum Drosophila revealed an impairment of learning and synaptic plasticity The 14-3-3 family of protein-binding proteins was first discovered in brain, where it comprises ∼1% of total soluble protein The multitude of functions exerted by 14-3-3 proteins is achieved through their ability to bind to phosphoserine/phosphothreonine–containing motifs of their ligands in a sequence specific manner. Two of the best known 14-3-3 consensus binding motifs are RSXpSXP and RXXXpSXP (pS represents the phosphorylated serine residue) in vivo. Moreover, our results demonstrate that, on the one hand, CKII supports binding of 14-3-3 to L1 and, on the other hand, 14-3-3 binding to L1 enhances L1 phosphorylation by CKII. Finally, expression of the mutated form of 14-3-3ζ, which impairs interactions of 14-3-3 with its binding partners, stimulated neurite elongation on an L1 substrate. Taken together, our data indicate that 14-3-3 proteins regulate biological functions of L1.For a better understanding of L1-mediated cell-cell interactions, it is crucial to elucidate the mechanisms of interaction between L1 and its intracellular binding partners To investigate a possible association between L1 and 14-3-3 proteins in mouse brain, immunoprecipitation experiments were performed. L1 was immunoprecipitated from brain membrane fractions of 3-week-old mice using an L1- specific antibody. A non-immune rabbit IgG was used as a negative control. The presence of 14-3-3 proteins in the resulting anti-L1 immune complex was analyzed using an anti-pan-14-3-3 mouse monoclonal antibody. As shown in 14-3-3ζ is one of the most abundantly expressed 14-3-3 isoform in neurons of the mammalian brain 1181, can be phosphorylated by CKII 2-3pS is a potential 14-3-3-binding motif 1181 was replaced by Ala to prevent L1 phosphorylation by CKII  and then subjected to CKII phosphorylation. At serial time points, the reaction was stopped by adding SDS-PAGE loading buffer. The effect of 14-3-3ζ on L1ICD phosphorylation was analyzed by loading the samples directly onto an SDS gel and then performing Western blotting using an anti-L1 monoclonal antibody. Phosphorylation of L1ICD was evident by the appearance of a second protein band, presumed to be phosphorylated L1, with slightly lower mobility in SDS-PAGE relative to non-phosphorylated L1ICD . An incr1181 has been implicated in normal endocytotic trafficking of L1 CKII-mediated phosphorylation of L1 at SerOur above-mentioned observations suggested a role for 14-3-3 in regulating L1 phosphorylation by CKII, putatively in endosomes. Considering the importance of endosomal trafficking for L1-mediated neurite growth in vivo, as demonstrated by coimmunoprecipitation. ELISA and pull-down experiments confirmed a direct interaction between 14-3-3ζ and the intracellular domain of L1 (L1ICD). We have also observed binding of recombinant 14-3-3β to L1ICD in vitro . However, the binding intensity was lower, suggesting that L1 may preferentially interact with specific 14-3-3 molecules. In the present study, we focused on the interaction between 14-3-3ζ and L1ICD. This interaction is enhanced by CKII-mediated phosphorylation of L1ICD. Notably, we found that 14-3-3ζ interacts not only with phosphorylated L1, but also with nonphosphorylated L1. Although most known 14-3-3-ligands possess phosphoserine- or phosphothreonine-based motifs, several interactions between 14-3-3 and nonphosphorylated motifs within ligand proteins have been described. Examples include the sequences VTPEER of the amyloid protein precursor (APP) ICD fragment In the present study 14-3-3 and L1 were found to associate 1181. Moreover, we found that Ser1181 is indispensable for the interaction of 14-3-3ζ with L1, as 14-3-3 failed to bind an S1181A L1ICD mutant in pull-down assays. This finding suggests that Ser1181 is also crucial for the interaction between 14-3-3 proteins and non-CKII-phosphorylated L1. At present we cannot completely exclude that additional phosphorylated or non-phosphorylated 14-3-3 binding sites exist in the L1ICD. Based on the “gatekeeper phosphorylation” concept The RSLESD sequence in the central part of the L1ICD, which, as discussed below, is the likely site of interaction for 14-3-3ζ, contains two acidic residues. These residues might mimic a phosphoryl group of consensus motifs and help to mediate the interaction of 14-3-3ζ with non-phosphorylated L1. Mutation of these two residues may be a suitable approach to clarify the mechanism of phospho-independent 14-3-3 binding to L1 in the future. The RSLESD sequence contains a RX2-3pS motif, which is a potential 14-3-3-binding site 1176), a residue adjacent to the RSLESD sequence essential for 14-3-3 binding, has been shown to prevent binding of L1 to the AP-2 adaptor protein, thereby blocking L1 endocytosis 1176, which could act as a regulatory signal. However, binding of L1 to CKII-phosphorylated 14-3-3ζ in vitro is not altered in the presence of AP-2 .As 14-3-3ζ interacts with nonphosphorylated and CKII-phosphorylated L1ICD, the question arose whether 14-3-3ζ utilizes the identified binding site (RSLESD) for phosphorylation-dependent and –independent interaction with L1. Notably, two previous studies showed that 14-3-3ζ interacts with phosphorylated and non-phosphorylated Tau protein and that two distinct 14-3-3 binding sites mediate these interactions 1181 by CKII was profoundly enhanced by 14-3-3ζ. One explanation is that L1 phosphorylation is promoted by initial interactions between 14-3-3ζ and (non-phosphorylated) L1ICD, resulting in L1ICD conformational changes and, consequently, increased susceptibility for CKII phosphorylation. It is known that 14-3-3 behaves like a molecular anvil, deforming its bound ligands while undergoing only minimal structural alterations itself The above-mentioned study on 14-3-3 - Tau interaction Nakata and Kamiguchi Given the importance of L1 trafficking for neurite extension, we also tested the influence of neuronally expressed 14-3-3ζ 1181. This, in turn, could inhibit targeting of L1 to late endosomes and lysosomes, resulting in an increased amount of L1 molecules on the cell surface. If more L1 molecules are available on the cell surface, this will stimulate the neuron's homophilic response to an L1 substrate, in accordance with our results. In line with such a model, an involvement of 14-3-3 proteins in the regulation of the subcellular localization of target proteins and the stabilization of targets at the cell surface has been demonstrated by several studies .E. coli. pDEST26-14-3-3ζ and pDEST26-14-3-3ζ K49E were transfected into mammalian cells for recombinant expression of His-14-3-3ζ or His-14-3-3ζ K49E, respectively. To enable prokaryotic expression of His-tagged L1ICD, including the amino acids Cys-Phe-Ile (CFI) of the transmembrane domain, the following steps were performed: RNA from murine brain was extracted with TRIzol according to the manufacturer's manual . The QuantiTect Rev. Transcription Kit was used for cDNA synthesis according to the manufacturer's protocol . L1 CFI was amplified from cDNA using gene specific primers and AccuPrime™ Taq DNA Polymerase (Invitrogen). The polymerase chain reaction (PCR) was started with 2 min at 94°C and followed by 30 cycles of 30 sec at 94°C, 30 sec at 55°C, and 60 sec at 68°C. All primers were synthesized by Metabion  based on the L1cam DNA sequence (NCBI accession number NM_008478.3). The PCR product was first ligated into pGEM-T Easy  using TA cloning, and subsequently subcloned into the expression vector pQE30 (Qiagen) using BamH I and Hind III. Plasmids containing single point mutations and the RSLESD deletion construct were prepared using the QuikChange II XL Site-Directed Mutagenesis Kit . All constructs were verified by DNA sequencing.pDEST15-14-3-3ζ, pDEST26-14-3-3ζ, and pDEST26-14-3-3ζ K49E were kindly provided by Dr. H. Fu, Emory University, Atlanta. pDEST15-14-3-3ζ was used for expression of GST-14-3-3ζ in Anti-14-3-3 (sc-1657), which recognizes all 14-3-3 isoforms according to the supplier's information, and anti-Rab 9 (sc-28573) antibodies were purchased from Santa Cruz Biotechnology , anti-14-3-3ζ (JP 18644) from IBL , anti-Rab 4 (610888) antibody from BD Transduction Laboratories  and the anti-GST antibody from GE Healthcare . Polyclonal antibodies against the mouse L1 extracellular domain were obtained by immunizing rabbits with a protein A-purified L1-Fc fusion protein consisting of the extracellular domain of mouse L1 and the Fc portion of human IgG E. coli BL21-AITM cells (Invitrogen) by L-arabinose induction. His-L1ICD, His-L1ICDS1181A, and His-L1ICDΔRSLESD were expressed in E. coli M15 cells by isopropyl-β-D-thiogalactoside induction and purified on Ni-NTA-agarose (Qiagen) and Ni-TED-agarose  according to the manufacturer's instructions. His-NCAM180 ICD l-lysine (PLL) was purchased from Sigma , human Fc was from Dianova .GST-14-3-3ζ fusion protein was expressed in 2, and 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF), Complete Protease Inhibitor Cocktail, EDTA-free , and Phosphatase Inhibitor Cocktail I (Sigma)). Brain homogenates were centrifuged at 17,000×g for 1 h. The 17,000×g supernatant thus obtained was centrifuged at 100,000×g for 1 h. The subsequent 100,000×g pellet was homogenized in 0.32 M sucrose in Tris buffer (50 mM Tris-HCl (pH 7.5), 1 mM CaCl2, and 1 mM MgCl2) and loaded onto a step gradient comprising layers of 2, 1.3, 1.1, 0.8, 0.5 and 0.25 M sucrose as described Brains from 7-day-old C57BL/6J mice were homogenized in homogenization buffer , 1 mM CaCl2, 1 mM MgCl2 in TBS-Tween (0.05% Tween)). Plates were washed 3 times with TBS-T and incubated for 1 h with anti-GST goat polyclonal antibody diluted 1∶4000 in TBST containing 1% BSA. After washing with TBST, wells were incubated with peroxidase-coupled secondary antibody in TBS-T containing 1% BSA, washed 4 times, and incubated with 0.1% ABTS substrate (Roche Diagnostics) in 100 mM acetate buffer, pH 5.0. Absorbances were measured at 405 nm. Correction for potential GST background signals was performed by subtracting absorbance values in wells incubated with GST only.Intracellular domains of L1 and NCAM180 (5 µg/ml) were immobilized overnight on a polyvinyl chloride surface  in TBS (10 mM Tris-HCl pH 7.4 and 150 mM NaCl). Wells were then blocked for 1 h with TBS containing 1% BSA and incubated for 1 h at RT with increasing concentrations of GST-14-3-3ζ (or GST-only control) diluted in buffer A , and 200 µM ATP with 2000 units/mL of CKII . The same assay was also performed with L1ICD proteins preincubated with GST or GST-14-3-3ζ at 4°C for 16 h. Where indicated, λ protein phosphatase 1  was added to aliquots of the CKII reaction mixture after 3 h, yielding a λPP1 concentration of 6000 units/mL. These aliquots were supplemented with 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM MnCl2, 2 mM DTT and 0.01% Brij 35, and incubated for 1 h at 30°C. To determine L1ICD phosphorylation, reaction aliquots were withdrawn at the indicated time points, mixed with an equal volume of SDS-PAGE sample buffer, and analyzed by SDS-PAGE and Western blotting. Blots were scanned and band intensities were determined.CKII mediated phosphorylation was investigated by incubating 15 µg of L1ICD proteins at 30°C for 3 h in a reaction mixture containing 20 mM Tris- HCl (pH 7.5), 50 mM KCl, 10 mM MgCl20 µg GST-14-3-3ζ fusion protein or an approximately equimolar amount of GST (10 µg) were coupled to 50 µl Glutathione Sepharose 4B beads (Sigma) for 2 h at 4°C, followed by preincubation with 5% BSA in pull-down buffer  for 2 h at 4°C. Beads were washed with pull-down buffer, and incubated with the total CKII phosphorylation reaction described above. Agarose beads were collected by centrifugation and washed extensively with 0.1% Nonidet P-40 pull-down buffer. Samples were analyzed by SDS-PAGE and Western blotting.3VO4, 1% Nonidet P-40, 0.5% SDS, 100 µM PMSF, Complete Protease Inhibitor Cocktail EDTAfree, and Phosphatase Inhibitor Cocktail I) were precleared using 25 µl of Protein A Agarose beads (Santa Cruz). Immunoprecipitation was performed with an anti-mouse L1 polyclonal antibody and as a negative control an nonimmune rabbit IgG. After 4 h of incubation, 25 µl of Protein A Agarose beads were added to the supernatant, and incubation continued overnight at 4°C. Protein A Agarose beads were collected by centrifugation, and washed 4 times with RIPA buffer. Bound proteins were eluted by boiling in SDS-PAGE sample buffer and subsequently analyzed by Western blotting using the indicated antibodies. Mouse brain vesicle fractions were lysed in lysis buffer  for 1 h at 4°C. Extracts were centrifuged at 21,000×g for 30 min at 4°C and the supernatants were further analyzed. Vesicle fractions containing 200 µg/ml protein in Protein A/G binding buffer  were precleared using 20 µl Protein G magnetic beads (Thermo Scientific) for 30 min at 4°C. Immunoprecipitation was performed with 5 µg anti-L1 antibody 557 and purified non-immune rat IgG as a control overnight at 4°C. Antibody-protein complexes were precipitated using 20 µl of Protein G magnetic beads. Samples were analyzed by SDS-PAGE and Western blotting.Mouse brain membrane fractions containing 1.5 mg/ml protein in modified RIPA buffer  without knowledge of substrate and vectors used for transfection. Statistical analyses were performed by Bonferroni's post-hoc comparison after two-way ANOVA with repeated measures (SigmaStat 3.5).A protocol for preparation, culture and transfection of primary rat embryonic E18 hippocampal neurons and determination of neurite length was adapted from


L1 retrotransposable elements are potent insertional mutagens responsible for the generation of genomic variation and diversification of mammalian genomes, but reliable estimates of the numbers of actively transposing L1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of L1 elements, several phylogenetic and L1Xplore analyses in the mouse genome suggest that 1,500–3,000 active L1 elements currently exist and that they are still expanding in the genome. Conversely, the human genome contains only 150 active L1 elements. In addition, there is a discrepancy among the nature and number of mouse L1 elements in L1Xplore and the mouse genome browser at the UCSC and in the literature. To date, the reason why a high copy number of active L1 elements exist in the mouse genome but not in the human genome is unknown, as are the potential mechanisms that are responsible for transcriptional activation of mouse L1 elements.F family of mouse L1 elements. The presence of sequence variations and polymorphisms in CpG islands of L1 promoters that arise from transition mutations indicates that CpG methylation could play a significant role in determining the activity of L1 elements in the mouse genome.We analyzed the promoter sequences of the 1,501 potentially active mouse L1 elements retrieved from the GenBank and L1Xplore databases and evaluated their transcription factors binding sites and CpG content. To this end, we found that a substantial number of mouse L1 elements contain altered transcription factor YY1 binding sites on their promoter sequences that are required for transcriptional initiation, suggesting that only a half of L1 elements are capable of being transcriptionally active. Furthermore, we present experimental evidence that previously unreported CpG islands exist in the promoters of the most active TA comprehensive analysis of mouse L1 promoters suggests that the number of transcriptionally active elements is significantly lower than the total number of full-length copies from the three active mouse L1 families. Like human L1 elements, the CpG islands and potentially the transcription factor YY1 binding sites are likely to be required for transcriptional initiation of mouse L1 elements. The long interspersed nuclear element-1 (LINE-1 or L1) is the most prolific class of mammalian retrotransposable elements, comprising 21 to 19% of the human and mouse genomic sequences L1 elements share the same organization and conserved motifs between mammalian species; a single line of successive L1 elements has been amplified between 40 and 12 million years in the primate lineage leading to humans F, GF, and A distinguished by their monomer sequences; younger TF <GF <A older) exist in the mouse genome and are active L1 is transcribed from its 5′-UTR internal promoter. Although mouse and human L1 ORFs are homologous, the promoter sequence contained within the 5′-UTR region shows no sequence homology between the two species A recent release of the L1Xplorer database Recent promoter analysis shows that both mouse and human L1 elements contain a putative E2F/Rb binding site (5′-G/CG/CCGGC-3′) within their 5′-UTR promoters F subfamily, together with the A and GF subfamilies are considered active elements F subfamily, which contains 875 elements, followed by 473 and 116 elements for the A and GF subfamilies, respectively. In addition, the mouse genome also contains 3 L1 elements of the oldest F subfamily (with highly truncated 5′-UTR promoters) and 34 unclassified L1 elements. These elements may not be currently active in the genome because their 5′-UTR sequences are heavily rearranged and divergent from one another beyond recognition.To identify and characterize potentially active L1 elements in the mouse genome, we utilized the non-redundant L1Xplorer (Ens24.33) database to ensure that our dataset contained only full-length, intact L1 elements including a 5′-UTR promoter, two open reading frames (ORF1 and ORF2), and 3′ poly-A tail sequences. Interrogating this database revealed the presence of 1,464 potentially active L1 elements residing in the mouse haploid genome . This figure supports previous estimates that the diploid mouse genome consists of approximately 3,000 potentially active L1 elements (2*1464 = 2928) F subfamily, followed by 4.26 and 4.1 monomers for A and TF subfamilies, respectively (Table S1). Given that only two monomers are sufficient for L1 promoter activity Because L1 promoter activity is thought to be proportional to the number of the monomers in that promoter, we next determined the average number of monomers present in each of the mouse L1's subfamilies. By characterizing the 5′-UTR promoters of the 1,464 potentially active L1 elements, we found an average of 5.6 monomers in the GL1 is an insertional mutagen capable of disrupting gene function as well as altering the regulatory properties and expression patterns of neighbouring genes. In humans, the highly active L1 elements residing within or close to known or predicted genes can affect the expression of nearby genes 2 = 0.346, p (two-tailed)  = 0.005). Statistical analysis of the distribution of L1 elements on the chromosomes is presented in Table S2. Although L1 elements are found in all the chromosomes, their abundance varies considerably. The highest density of potentially active L1 elements resides in the X-chromosome (average ratio 1.81%), whereas the autosomes contain lower densities of L1 elements (with ratios between 0.24% and 1.07%). Consistent with other species, the X-chromosome in mice seems disproportionately enriched for intact L1 elements as compared to autosomes, indicating an L1 insertional bias; however, natural selection may also be the driving force. As suggested by other studies in humans, and DrosophilaTo identify the density of L1 elements within and near genes, we initially determined how many L1 elements are present on each mouse chromosome. As expected, L1 elements are present on every chromosome and the distributions of L1 subfamilies are shown in  and Table S3). The oldest inactive F subfamily and unclassified L1 elements were also found in intergenic regions. Analyzing these 953 L1 element locations and nucleotide sequences around the L1 insertion sites revealed a significantly high AT-rich content . This could partly explain why the intergenic regions of the mouse genome contain lower gene density and higher accumulation of L1 elements that prefer AT-rich regions for their insertion. Activation of L1 elements from the AT-rich intergenic regions might lead to accumulation of L1 elements within this gene-poor region of the genome, but the probability of L1 elements interfering with genes would be quite low. Because the intergenic genomic sequences are mostly bundled into repressive heterochromatin To explore the relationship between the presence of L1 elements and neighboring gene expression, we next looked in more detail at the distribution of L1 elements by locating all elements in the mouse genome relative to annotated genes. Using a Perl script, we estimated how many neighboring genes exist within flanking sequences 100-kb in either direction of potentially active L1 elements. Surprisingly, we found that 64% (n = 953) of the intact L1 elements occupy intergenic regions within the 100-kb regions; that is, they are not within exons, introns, or untranslated regions  subfamilies there are neighboring genes within a 100-kb window of the L1 elements; their chromosomal distributions are indicated in 2 = 6.688, dt = 1, p = 0.009). Consistent with previous estimates Table S4). This uneven distribution of L1 elements in the proximity of genes prompted us to ask whether the trend in L1 frequency with respect to genes is due to surrounding GC content, or whether genes exert independent effects on the distribution of L1 density. Analysis of the GC-content across 20-kb surrounding regions of L1 elements did not reveal high significant difference from what would be expected by chance in regions surrounded by genes . At present, it is unclear why some gene-rich regions, but not others are prone to L1 element insertion.Remarkably, for up to 36% of TOne possible hypothesis is that the local chromatin environment and associated DNA sequences might influence the density of L1 elements in some genomic regions of mouse chromosomes. Recently, Graham and Boissinot proposed that the transcriptional status at the insertional site could favour the accumulation of L1 inserts near genes Table S5). The proportion of genes oriented in the opposite (antisense) transcriptional direction with respect to L1 elements was roughly three times greater than the same (sense) orientation over the 100-kb regions . Of the genes located within 100-kb of an L1 element, the majority  are located more than 20-kb from an L1 element, suggesting that L1 elements are preferentially landed at significant distances from genes , followed by the A subfamily . The overrepresentation of TF L1 elements indicates that these regions are probably prone to L1 integration. Because L1 insertions in these regions are generally more than 20 kb away from genes, we hypothesize that negative selection pressure against deleterious effects of L1 insertion could also play a role in the genomic distribution of L1 elements. Surprisingly, many of the genes identified within 100-kb of an L1 element are tissue-specific genes, expressed mainly in testis, placenta, and brain tissues, as well as in neural progenitor cells (S6).To investigate whether such a relationship exists in the genomic distribution of mouse L1 elements, we determined the orientation and type of the RefSeq genes located upstream and downstream of each L1 subfamily . The gene expression and functional genomics dataset (www.ebi.ac.uk/microarray-as/ae/) suggests that these genes are expressed exclusively in testis and brain during development. Because of the presence of potentially active L1 elements near the genes, it is tempting to speculate that the activity of L1 elements may affect the expression patterns of these developmentally transcribed genes. As demonstrated by human transcriptome studies Arabidopsis transposons in which the expression of the flowering-time gene FWA is affected by the methylation status of nearby transposable elements Intriguingly, we found that three genes  reside within a 5-kb of an L1 element and are oriented in the same transcriptional direction as the L1 element . Using the TRANSFAC database, we searched the repeating monomer region of the 953 L1 promoters representing intergenic L1 elements and the 546 L1 promoters representing L1 elements with neighboring genes for presence of the conserved RUNX3 and YY1 binding sites. A previous study in human L1 promoters identified a potential RUNX3-binding site (5′-TGCATTTCCATCTGAGGTA-3′) starting at base pair +806 to +824 upstream of ATG start site To analyse transcription factor binding motifs, we extracted all the L1 promoter sequences from 1,464 potentially active T5′-GGTCGCCATCTTGGT-3′. Comparative analysis of both the human and mouse L1 promoters containing putative RUNX3 and YY1 binding sites are shown in Table S7). There are 819 promoters in the TF subfamily followed by 427 in the A subfamily and 113 in the GF subfamily matching this criteria. Of the 819 TF promoters, 290 have neighboring genes and the remaining 529 represent intergenic regions. By aligning the sequences of TF monomers with the consensus sequence of the YY1 motifs, we found that 17% (48/290) of TF elements from gene-rich regions and 11% of TF elements (58/529) from intergenic regions contain mutations within the putative YY1 binding site of a ‘minimal’ promoter that is composed of only two monomers (Table S7). Consistent with this assumption, previous studies of human L1 promoters also identified the YY1-binding site as an important sequence for L1 expression and found that mutations in the putative YY1 motif markedly disrupt promoter activity F promoters (242/290) reside close to RefSeq genes and contain potentially functional YY1 motifs in an array of more than two monomers on the same promoter, indicating that these elements are potentially capable of influencing the expression of neighboring genes.Interestingly, the TRANSFEC analysis of mouse L1 promoters showed potential YY1 binding sites within their monomers. The consensus-binding site for the transcription factor YY1 was identified as monomers Figure 5F subfamilies that have neighboring genes also contain highly degenerated YY1 motifs in all monomers of the promoters (differing by up to 55% from the consensus sequences); we therefore omitted these elements from further analysis. Only a small subset of A and GF elements from intergenic regions (24 elements) still contain putative YY1 motifs, suggesting that only these are capable of activity. This may explain why functionally active A and GF elements are rare in the mouse genome. Consistent with this observation, no GF subfamily of L1 elements have been detected in any Mus spretus or Mus musculus genomes except the genome of laboratory strain 129/Sv F subfamily L1 elements from an embryonic stem cell library of strain 129/Sv, it was previously estimated that 900 A and 400 GF elements are active in the mouse genome F elements in the mouse genome. Nevertheless, at least three L1 elements belonging to the A-type subfamily have recently been shown to be active in mouse vascular smooth muscle cells F subfamily L1 elements could be still active in some specialized cell types. Taken together, the data presented in our study suggest that the vast majority of TF promoters contain YY1 motifs within their monomer regions and that this may have implications for transcriptional initiation of the mouse L1 elements.Unexpectedly, most L1 promoters in the A and GThe mammalian L1 regulatory sequences, though not homologous, share several features with viral and housekeeping promoters; they contain CpG islands and lack the traditional TATA boxes found in cell-specific PolII promoters To investigate this, we extracted the sequences of the 1,464 L1 promoters in the mouse genome and searched for CpG Islands using the EMBOSS CpGPlot analysis. CpG islands were defined as DNA sequences longer than 200 bp with >50% GC content and an observed/expected presence of CpG >0.6. By analysing the entire promoter sequences, we identified 124 mouse L1 elements that contain CpG islands in their promoter regions. This represents ∼8.5% of the estimated genomic copies of L1 in the mouse genome, much lower than that of the human and rat L1 elements where the majority contain CpG islands , followed by 12% (15/124) and 7% (9/124) in the older GF and A-subfamilies, respectively  within a distance of 5 kb, it is tempting to speculate that the methylation status of L1 CpG islands might be associated with expression of developmentally transcribed genes during the process of cellular development.Remarkably, 81% of CpG islands are present in the highly active, young Tectively Figure 8F L1 elements.CpG islands are selectively associated with the regulatory regions of L1 elements and are generally methylated in somatic cells of humans and rats. In fact, their methylation status has been reported to prevent constitutive expression of L1 elements F promoters (IDs: 711 and 837) from the X-chromosome and cloned them into the upstream region of the luciferase reporter gene. BLAST analysis of the promoter sequences showed that both promoters were homologous and almost identical in terms of the size, number of TF monomers and the position of YY1 and E2F binding sites, however only one of the promoters contained a CpG island (that is ID: 711). To test whether these promoters were able to initiate transcription, we transfected mouse NIH3T3 cells with luciferase reporter constructs driven by either a CpG or non-CpG L1 promoter and performed a dual-luciferase assay with the Renilla luciferase plasmid (p<0.001). As expected, luciferase expression was barely detectable in vector alone-transfected control cells. This finding suggests that CpG islands in mouse L1 elements are part of a promoter regulatory region that is required for the elevated expression of L1 elements in mouse cells. Consistent with this study, a previous report of rat L1 elements also revealed that L1 activation occurs only when the promoter contains a CpG island at its 5′-UTR region To do this, we isolated the complete sequences of two TF promoters (IDs: 711 and 837) contain intact YY1 binding sites except for the presence or absence of a CpG islands, we next determined whether the presence of the YY1 transcriptional factors binding sites within the CpG islands is required for L1 promoter activity. To do this, we isolated two additional TF promoters (IDs: 1412 and 1457), which contained mutations in the YY1 motifs within a CpG island, and cloned them into luciferase reporter constructs. By measuring the luciferase activities, we found that the promoter activities of these mutated YY1 sites decreased by two-fold (p<0.001) compared with the intact YY1 motifs within the CpG islands (ID: 711). On the other hand, the TF promoter (ID: 1312) that contained both YY1 mutations and an absence of CpG islands did not express any luciferase at all. Together, this data clearly indicates that, like humans, CpG islands in L1 promoters may play an important role in the transcriptional activation of mouse L1 elements and that the presence of CpG islands most likely influences the ability of YY1 motifs to act in transcriptional initiation of promoters. Further studies are required to identify the critical CpG sites and to conform the roles and functional significance of YY1 motifs within a CpG island of mouse L1 promoter. Nonetheless, our study show that only a half of mouse L1 elements  are capable of activity–a significantly lower than we initially estimated. Of the 710 mouse L1 elements, only 124 contain previously unreported CpG islands in their promoters that showed a high level of promoter activity and that there is a difference in the level of expression with and without a CpG island.Because both Thttp://l1base.molgen.mpg.de/). L1 elements devoid of intact 5′-UTRs, ORFs, and 3′-UTR sequences were removed from the dataset to ensure that it contained only full-length, potentially active L1 elements (n = 1467). We mapped the genomic locations of all the L1 elements with the UCSC mouse genome assembly  using BLAT analysis, with data accuracy greater than 0.98. Classification of L1 subfamilies was carried out using the RepeatMasker program (provided by Ensembl) and a customised version of the monomer search modules of L1Xplorer Sequences and annotation data for mouse L1 elements were retrieved from the L1Base  database (http://genome.ucsc.edu/) and analyzed the position and location of L1 elements in the RefSeq gene annotations using a Perl script. For each L1 element, we analyzed the genomic regions extending 100-kb upstream of the L1 start position and 100-kb downstream of the L1 end position. Sense and antisense orientation of genes were defined relative to the L1 element being analysed; genes orientated in the same direction as the L1 element are designated “sense” and genes in the opposite direction are designated as “antisense.” The nearest gene adjacent to each L1 element and the distance from the gene to the L1 element was recorded. To confirm the gene annotation, a BLAST query was performed separately against the RefSeq cDNA database. For genes with no available functional annotations, we performed a search against the NCBI non-redundant or FANTOM3 databases to identify any homologous genes with functional annotations. Genes with no functional annotations were classified as “hypothetical genes” if they encoded an unidentified protein with more than 100 amino acids.We downloaded the RefSeq database of the mouse genome  from the UCSC Genome Browser . For identification of transcriptional factor binding site motifs, each promoter was searched by using TRANSFAC  (http://www.gene-regulation.com/pub/databases.html) with a cut-off value >0.9. The distances between the matches and the ATG start site were calculated and the number of matches in every monomer from the ATG site was plotted.The promoter sequences of L1 elements from L1Xplorer or the UCSC genome database were extracted using a suite of Perl scripts that detect and extract the core L1 promoter sequences by performing BLAST searches. The accession numbers of the L1 promoter are provided in 5′-TTCCTCGAGCCCAGAATAACAATCATCCA-3′; 837FOR, 5′-TTCCTCGAGGCAGACAACCTTACGTTATG-3′; 1412FOR: 5′-TTCCTCGAGTCCTCTTGGGTGAATTTTCTTC-3′; 1457 FOR: 5′-TTCCTCGAGAGATGGGGACACACACATCC-3′; 1312FOR: 5′-TTCCTCGAGGGGTGTTTAGTAACCATGTCTGG-3′ and REV: 5′-AATAAGCTTCTGGTAATCTCTGGAGTTAGTAGT-3′). All plasmids with correct inserts were confirmed by sequencing from both ends to ensure that the correct sequence was cloned. A Renilla luciferase vector, pRL-CMV (Promega) was used to correct the differences in transfection efficiency. Mouse NIH3T3 fibroblast cells were cotransfected with the modified pGL3 firefly luciferase (under the control of each TF promoter) and the Renilla luciferase reporter plasmid. Firefly and Renilla luciferase assays were measured after 48 h with the Dual-Luciferase Reporter assay system (Promega). Firefly activity was normalized to Renilla activity, as described previously A full-length promoter of TF elements was amplified by PCR from mouse genomic DNA and cloned into the reporter plasmid pGL3 (Promega) upstream of the luciferase gene Click here for additional data file.Table S2The distribution of L1 elements in mouse chromosomes and the statistical analysis of correlations between chromosome size and L1 density.(0.10 MB PDF)Click here for additional data file.Table S3The distribution of L1 elements in intergenic regions in mouse chromosomes.(0.04 MB PDF)Click here for additional data file.Table S4The distribution of L1 elements with neighboring genes in mouse chromosomes.(0.04 MB PDF)Click here for additional data file.Table S5The list of L1s' neighbouring genes and their locations.(0.09 MB PDF)Click here for additional data file.Table S6List of tissue-specific genes in the vicinity of L1 elements within 20-kb distance.(0.02 MB PDF)Click here for additional data file.Table S7The mutation rates at the YY1 transcription binding sites in Tf subfamily.(0.01 MB PDF)Click here for additional data file.