NANOG, a multipotency marker, and a notable decrease in HOX gene expression in LAB-incorporated cells. During the cell culture, the LAB-incorporated cell clusters stop cell division and start to express early senescence markers without cell death. Thus, LAB-incorporated cell clusters have potentially wide-ranging implications for cell generation, reprogramming, and cell-based therapy.The human gastrointestinal tract is colonized by a vast community of symbionts and commensals. Lactic acid bacteria (LAB) form a group of related, low-GC-content, gram-positive bacteria that are considered to offer a number of probiotic benefits to general health. While the role of LAB in gastrointestinal microecology has been the subject of extensive study, little is known about how commensal prokaryotic organisms directly influence eukaryotic cells. Here, we demonstrate the generation of multipotential cells from adult human dermal fibroblast cells by incorporating LAB. LAB-incorporated cell clusters are similar to embryoid bodies derived from embryonic stem cells and can differentiate into endodermal, mesodermal, and ectodermal cells in vivo and in vitro. LAB-incorporated cell clusters express a set of genes associated with multipotency, and microarray analysis indicates a remarkable increase of  Living organisms have been classified on the basis of cell structure into two groups: the eukaryotes and the prokaryotes. However, genomic analyses have shown that bacteria can be as widely divergent in their evolutionary history as any prokaryote is from any eukaryote. The prokaryotes comprise two distinct groups that are called the eubacteria and the archaebacteria. Thus, the living world has three major divisions: eubacteria, archaebacteria, and eukaryotes Humans are in contact with components of the microflora from birth. Thus, a delicate balance exists in the symbiotic relationship between microorganisms and the human host during metabolic activities. When considering the positive influence of microorganisms on eukaryotic cells, lactic acid bacteria (LAB) deserve to be studied. The LAB form a group of related, low-GC-content, gram-positive bacteria and occupy important niches in the gastrointestinal tract of humans NANOG in LAB-incorporated cells than iPS cells. The LAB-incorporated cell clusters stopped proliferating and expressed early senescence markers during culture. Gene expression profiling indicated a remarkable remodeling of HDFs, especially increased NANOG and a notable decrease in HOX genes, showing that the normal skin cells acquired the multipotency. This study establishes LAB-incorporated cell clusters as an accessible human multipotential cell type for a better understanding of the molecular mechanism of cell origins and reprograming.Thus, we studied the reaction of human fibroblasts, which are far different from digestive cells, when incorporated with LAB. To determine whether LAB can directly affect human gene expression in general, we incorporated LAB into adult human dermal fibroblasts (HDFs) and observed cell clusters similar to embryoid bodies derived from embryonic stem cells. The LAB-incorporated cell clusters expressed a set of genes associated with multipotency and differentiated into cell types of all three germ layers both in vivo and in vitro. Our quantitative RT-PCR analysis showed the higher expression of Lactobacillus acidophilus; JCM 1021) into adult HDFs that were pretreated with trypsin/EDTA to remove their cell surface proteins. After 2–3 days incubation, cell clusters  were generated similar to the embryoid bodies formed by human embryonic stem cells (hESCs) at an early stage  for the incorporation, they also demonstrated the ability to generate cell clusters  failed to form cell clusters (data not shown). Most of these cell clusters were attached to the dish and looked similar to those of hESC-derived embryoid bodies and/or Muse cells isolated from stress-tolerant mesenchymal cell populations Lactobacillus acidophilus; JCM 1021)  –C. The LCM 1021) .NANOG, SOX2, OCT3/4, and TDGF1, but not GDF3, FGF4, REX1, or ECAT15, while iPS cells expressed all of these markers. To compare the cell characteristic, the mRNA expression levels of NANOG, OCT4, and SOX2 of LAB-incorporated cell clusters at 14 days after incorporation and iPS cells were examined by quantitative RT-PCR , and microtubule-associated protein-2  in LAB-incorporated cell clusters that were cultured in the differentiation medium , each containing 8–11 genes. Interestingly, 21 HOX genes were downregulated in LAB-incorporated cell clusters by a factor of 15 or more among 50 genes that had a GO term of “development”  convert normal skin cells into multipotent cells.This study describes the generation of multipotent cells by incorporating LAB into adult HDFs. The LAB-incorporated cell clusters can differentiate into endodermal, ectodermal, and mesodermal cells in vivo and in vitro. Furthermore, the LAB-incorporated cell clusters are positive for a set of genes associated with multipotency and ALP staining. Moreover, the LAB-incorporated cell clusters stop dividing, retaining the cluster structure, and express early senescence markers to exhibit cell differentiation ability. Finally, the microarray analysis demonstrates a notable decrease in The microbial colonization of the human gastrointestinal tract is an evolution-driven process that has beneficial effects on immune function, nutrient processing, and a broad range of the host activities. Mucus is secreted by the epithelial surfaces throughout the gastrointestinal tract and provides a protective barrier between the underlying epithelium and the lumen, which contains noxious agents, destructive hydrolases, and microorganisms NANOG, a gene related to pluripotency, and the downregulation of HOX genes, which are associated with early development. NANOG is one of the canonical quartets of transcription factors employed to reprogram human fibroblasts and dose-facilitate molecular reprogramming NANOG is expressed in pluripotent embryonic cells and derivative ES cells, and forced NANOG expression is sufficient to drive the cytokine-independent self-renewal of undifferentiated ES cells HOX genes encode DNA-binding proteins that regulate gene expression and control various aspects of early embryonic development HOX genes are divided into two broad categories: a family of 38 genes that are related to the Antennapedia gene of Drosophila species, and the other group comprises more distally related genes. These data suggest that LAB-incorporated cell clusters lose their positional information and revert to the pluripotent state. Although many questions regarding the actual molecular processes occurring in LAB-incorporated cell clusters remain unanswered, further epigenetic studies will be helpful to characterize LAB-incorporated cell clusters.Gene expression profiles in LAB-incorporated cell clusters demonstrated two interesting issues: the upregulation of Pluripotent stem cells are able to differentiate into all types of cells in the body and have great potential for applications in regenerative medicine, developmental biology research, and drug development. ES cells, which are derived from the inner cell mass of the blastocyst, can differentiate into more than 200 types of cells. However, the fact that the isolation and establishment of human ES cell lines involve embryo destruction leads to extensive ethical concerns and debates. iPS cells, from fibroblasts with four Yamanaka factors, share a similar gene expression profile and differentiation potential with ES cells Lactobacillus acidophilus (JCM 1021), Streptococcus salivarius subsp. thermophilus (JCM 20026), Lactobacillus sp. (JCM 20061), and Lactococcus lactis subsp. lactis (JCM 20101). E. coli  were grown in LB medium and prepared for incorporation. LABs were grown in M.R.S. medium (OXOID). After treatment of HDFs with 0.1% trypsin and 1 mM EDTA for 5 min at 37°C, 5×105 cells suspended in 2 ml of Fibroblast Growth Medium (without gentamycin) and approximately 1×108 LAB suspended in 60 µl of Fibroblast Growth Medium (without gentamycin) were plated into a six-well dish (NUNC) containing 10 µg/ml lactoferrin (Sigma). After 5 days of incubation, half of the medium (1 ml) was replaced with Fibroblast Growth Medium containing 0.5×108 LAB. The medium was changed every 3 days, and the clusters were collected at the indicated times. For longer culture, LAB-incorporated cell clusters after 8 days of incorporation were maintained in human ES cell culture medium .HDFs from the facial dermis of a 34-year-old Caucasian female were purchased from Cell Applications, Inc. We used the HDFs for the generation of LAB-incorporated cell clusters within three passages after receipt. The HDFs were maintained in Fibroblast Growth Medium  containing 0.1% gentamycin (Sigma). We purchased the following LAB strains from RIKEN BioResource Center: 2, and 40 mM citric acid and then titrated with NaH2PO4 to pH 6.0.The LAB-incorporated cell clusters at 12 days after incorporation were washed twice in PBS, fixed for 10 min in 4% PFA, and embedded in Optimal Cutting Temperature compound (Sakura). Sections (12 µm) were incubated for 12 h at 37°C with freshly prepared ß-gal staining solution containing 1 mg/ml 5-bromo-4-chloro-3indolyl-β-D-galactopyranoside , 5 mM potassium ferrocyanide, 150 mM NaCl, 2 mM MgClFor immunocytochemistry, LAB-incorporated cell clusters were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). After washing with PBS, the cells were treated with PBS containing 5% heat-inactivated goat serum and 0.1% Triton X-100 for 30 min at RT. The following primary antibodies were used for immunocytochemistry: rabbit anti-NANOG (ReproCELL), mouse anti-Oct-3/4 (Santa Cruz Biotechnology), rabbit anti-SOX2 (Millipore), rat anti-SSEA-3 (Millipore), mouse anti-SSEA-4 (Millipore), mouse anti-TRA-1-81 (Millipore), mouse anti-vimentin (Santa Cruz), rabbit anti-desmin (Thermo), mouse anti-Tuj1 (DSHB), and mouse anti-neurofilament (ZYMED). After three washes with PBS, the sections were incubated with anti-mouse IgG antibody conjugated with Cy3 or FITC (Jackson ImmunoResearch), anti-rabbit IgG antibody conjugated with Cy3 (Amersham), or anti-rat IgG or IgM antibody conjugated with Cy3 (Jackson ImmunoResearch) in the presence of DAPI in the antibody dilutions for 2 h at RT.For neural induction with floating, LAB-incorporated cell clusters were cultured for 14 days in DMEM/F12 medium containing B-27 supplement (GIBCO), 1% FCS, and 20 ng/ml bFGF (Sigma). LAB-incorporated cell clusters were cultured in the floating condition for 14 days in DMEM/F12 medium containing 10% FCS to induce cell differentiation. For cell differentiation induction under the attachment condition, LAB-incorporated cell clusters were cultured on PLL-laminin-coated coverslips for 14 days in DMEM/F12 medium containing 1% FCS and 20 ng/ml bFGF (Sigma). The following primary antibodies were used for immunocytochemistry: mouse anti-vimentin (Santa Cruz), rabbit anti-desmin (Thermo), mouse anti-Tuj1 (DSHB), rabbit anti-α-SMA (Thermo), mouse anti-α-fetoprotein (DAKO), rabbit anti-GFAP (DAKO), and mouse anti-neurofilament (ZYMED). After three washes with PBS, the sections (10 µm) were incubated with anti-mouse IgG antibody conjugated with Cy3 (Jackson ImmunoResearch) or anti-rabbit IgG antibody conjugated with Cy3 (Amersham) in the presence of DAPI in the antibody dilutions for 2 h at RT.For the lineage-specific differentiation, the LAB-incorporated cell clusters were cultured for 3 weeks in STEMPRO Adipogenesis, Chondrogenesis, and Osteogenesis Differentiation Medium (GIBCO). The culture media were changed every 3 days. Adipocytes were identified by the production of lipid droplets, detected by staining with Oil Red O. Cells were washed with PBS and overlaid with 0.18% Oil Red O dye/60% 2-propanol (Sigma) solution for 15 min, then rinsed in distilled water. Chondrocytes were revealed by Alcian Blue staining, which detects the synthesis of glycosaminoglycans. Cells were fixed with 4% PFA for 30 min and washed three times with PBS, then incubated with 1% Alcian Blue solution (Wako) in 3% acetic acid at pH 2.5 for 30 min. Osteogenic differentiation, indicated by calcium deposits, was revealed by Alizarin Red S staining. Cells were fixed with 4% PFA for 30 min. After rinsing in distilled water, cells were stained with 40 mM Alizarin Red S (Sigma) solution at pH 4.2 and rinsed in distilled water.We have purchased iPS (IPS-TIG120-3f7) from Center for iPS Cell Research and Application . Total RNA of iPS cells and LAB-incorporated cells at 6 or 12 days after incorporation was purified using Trizol reagent (Invitrogen) and then treated with DNase I (Invitrogen) to remove genomic DNA contamination. The first-strand cDNA was synthesized from 1 µg total RNA using an oligo d(T)12–18 primer and Superscript III reverse transcriptase (Invitrogen). The PCR was performed with the following primers:5′-TGATGCATAGCCGAGTTGAG-3′);16S ribosomal RNA-S (5′-CAATAAATCCGGACAACGCT-3′);16S ribosomal RNA-AS (5′-GACAGGGGGAGGGGAGGAGCTAGG-3′);OCT3/4-S (5′-CTTCCCTCCAACCAGTTGCCCCAAAC-3′);OCT3/4-AS (5′-GGGAAATGGGAGGGGTGCAAAAGAGG-3′);SOX2-S (5′-TTGCGTGAGTGTGGATGGGATTGGTG-3′);SOX2-AS (5′-CAGCCCCGATTCTTCCACCAGTCCC-3′);NANOG-S (5′-CGGAAGATTCCCAGTCGGGTTCACC-3′);NANOG-AS (5′-GCGTCCTGGGAAGGGAGATCCGGAGC-3′);MYC-S (5′-TTGAGGGGCATCGTCGCGGGAGGCTG-3′);MYC-AS (5′-CTGCTGCCTGAATGGGGGAACCTGC-3′);TDGF1-S (5′-GCCACGAGGTGCTCATCCATCACAAGG-3′);TDGF1-AS (5′-CAGATC CTA AAC AGC TCG CAG AAT-3′);REX1-S (5′-GCGTACGCAAATTAAAGTCCAGA-3′);REX1-AS (5′-CTACAACGCCTACGAGTCCTACA-3′):FGF4-S (5′-GTTGCACCAGAAAAGTCAGAGTTG-3′);FGF4-AS (5′-CTTATGCTACGTAAAGGAGCTGGG-3′);GDF3-S (5′-GTGCCAACCCAGGTCCCGGAAGTT-3′);GDF3-AS (5′-GGAGCCGCCTGCCCTGGAAAATTC-3′);ECAT15-1-S (5′-TTTTTCCTGATATTCTATTCCCAT-3′);ECAT15-1-AS (5′-GCGGGGACTAGTGGAGAAG-3′);P15-S (5′-CTGCCCATCATCATGACCT-3′);P15-AS (5′-CAACGCACCGAATAGTTACG-3′);P16-S (5′-CTGCCCATCATCATGACCT-3′);P16-AS (5′-CTACTGAGGAGCCAGCGTCT-3′);ARF-S (5′-CTGCCCATCATCATGACCT-3′).ARF-AS  were treated with PBS containing 5% heat-inactivated goat serum and 0.1% Triton X-100 for 30 min at RT. The following primary antibodies were used for immunohistochemistry: mouse anti-human mitochondria (abcam), rabbit anti-α-SMA (Thermo), mouse anti-α-fetoprotein (DAKO), and mouse anti-neurofilament (ZYMED). The sections were incubated with anti-mouse IgG antibody conjugated with Cy3 or FITC (Jackson ImmunoResearch) or anti-rabbit IgG antibody conjugated with Cy3 (Amersham) in the presence of DAPI in the antibody dilutions for 2 h at RT.Total RNA from the HDFs and LAB-incorporated cell clusters was labeled with Cy3 (2 replicates). Each sample was hybridized once using a one-color protocol. Sample labeling and hybridization to Whole Human Genome Microarray 4×44K (Agilent) were performed by Oncomics (Japan). The arrays were scanned using a G2505B Microarray Scanner System (Agilent), and the Affymetrix data were analyzed by using GeneSpring GX11.5.1 software (Agilent).Figure S1Typical LAB-incorporated cell clusters generated by HDFs and LAB. (A) The cell clusters were not generated from HDFs without LAB that were dissociated with trypsin/EDTA. (B) Lactobacillus acidophilus (JCM 1021) is able to generate LAB-incorporated cell clusters from HDFs that were dissociated with trypsin. (C) Lactococcus lactis subsp. lactis (JCM 20101), Streptococcus salivarius subsp. thermophilus (JCM 20026), and Lactobacillus sp. (JCM 20061) lactic acid bacteria are also able to generate LAB-incorporated cell clusters from HDFs. Scale bars, 100 µm.(PDF)Click here for additional data file.Figure S2LAB-incorporated cell clusters are ALP positive. (A) LAB-incorporated cell clusters generated by Lactobacillus acidophilus (JCM 1021) from HDFs are ALP positive. (B) LAB-incorporated cell clusters generated by Lactococcus lactis subsp. lactis (JCM 20101), Streptococcus salivarius subsp. thermophilus (JCM 20026), or Lactobacillus sp. (JCM 20061) from HDFs are ALP positive. Scale bars, 100 µm.(PDF)Click here for additional data file.Figure S3Generation of GFP-MEF-derived LAB-incorporated cell clusters. Lactic acid bacteria  can generate cell clusters from LAB-incorporated GFP-MEFs. Lower  and higher (C) magnifications of LAB-incorporated GFP-MEF clusters after 6 days of incorporation. (D) LAB-incorporated GFP-MEF clusters are ALP positive.  Bright field.  GFP. Scale bars: 500 µm in , 100 µm in . For GFP-MEF preparation, the uteri isolated from 12.5-day-pregnant GFP-mice (C57BL/6-Tg(CAG-EGFP)C15-001-FJ001Osb mice) were washed with PBS. The head and visceral tissues were removed from the isolated embryos. The remaining bodies were washed, minced using scissors, transferred into a 0.1% trypsin/1 mM EDTA solution, and incubated at 37°C for 20 min. An equal amount of culture medium was added and pipetted up and down a few times to dissociate tissue. After passing through a cell strainer , cells were collected by centrifugation  and cultured in a 10 cm dish . A total of 5×105 cells (2 ml) and approximately 1×108 lactic acid bacteria suspended in 60 µl Fibroblast Growth Medium containing 10 µg/ml lactoferrin (Sigma) were plated into the 6-well dish (NUNC).(PDF)Click here for additional data file.Figure S4Double immunostaining. To examine the heterogeneous expression pattern of pluripotency markers in LAB-incorporated cell clusters at 14 days after incorporation, double staining was performed with rabbit anti-NANOG (ReproCELL) and mouse anti-OCT3/4 (Santa Cruz Biotechnology) antibodies. After three washes with PBS, the sections (10 µm) were incubated with anti-mouse IgG antibody conjugated with Cy3 and anti-rabbit IgG antibody conjugated with FITC in the presence of DAPI in the antibody dilutions for 2 h at RT. The long white arrows indicate cells that express NANOG and OCT3/4 in the nucleus. The short arrow indicates the cell that expresses only NANOG. The large arrowhead indicates the cell that expresses OCT3/4 in the cytoplasm. The small arrowhead indicates the cell that expresses both NANOG and OCT3/4 in the cytoplasm. Scale bar: 100 µm in (A–D), 18 µm in (E–H).(PDF)Click here for additional data file.Figure S5RT-PCR analysis. RT-PCR analysis of native HDFs, LAB-incorporated cell clusters, and LAB-incorporated cell clusters induced to differentiate in culture medium (DMEM/F12 medium containing 10% FCS) for 12 days. Note that LAB-incorporated cell clusters induced to differentiate express α-fetoprotein and GATA6. The PCR was performed with the following primers: α-fetoprotein-S (5′-CCACTTGTTGCCAACTCAGTGA -3′); α-fetoprotein-AS (5′-TGCAGGAGGGACATATGTTTCA-3′); GATA6-S (5′-CCTGCGGGCTCTACAGCAAGATGAAC-3′); GATA6-AS (5′-CGCCCCTGAGGCTGTAGGTTGTGTT-3′); MAP-2-S (5′-ACTACCAGTTTCACACCCCCTTT-3′); MAP-2-AS (5′-AAGGGTGCAGGAGACACAGATAC-3′).(PDF)Click here for additional data file.Figure S6Cell differentiation. LAB-incorporated GFP-MEF clusters were cultured on PLL-laminin-coated coverslips for 14 days in DMEM/F12 medium containing 1% FCS and 20 ng/ml bFGF (Sigma). The following primary antibodies were used for immunocytochemistry: rabbit anti-desmin (Thermo), mouse anti-Tuj1 (DSHB), rabbit anti-α-SMA (Thermo), and rabbit anti-GFAP (DAKO). After three washes with PBS, the slides were incubated with anti-mouse IgG antibody conjugated with Cy3 (Jackson ImmunoResearch) or anti-rabbit IgG antibody conjugated with Cy3 (Amersham) in the presence of DAPI in the antibody dilutions for 2 h at RT. LAB-incorporated GFP-MEF clusters after 14 days of incorporation were cultured for 3 weeks in STEMPRO Adipogenesis and Osteogenesis Differentiation Medium (GIBCO). Adipocytes were identified by the production of lipid droplets, detected by staining with Oil Red O. Osteogenic differentiation, indicated by calcium deposits, was revealed by Alizarin Red S staining. Scale bars: 20 µm in (A–D), 20 µm in .(PDF)Click here for additional data file.Figure S7Time course of the differentiation of LAB-incorporated cells. LAB-incorporated cell clusters after 14 days of incorporation were treated to the conditions that induced differentiation into adipocytes, osteocytes, or chondrocytes. The cell clusters were crumbled into the single cell and differentiate into adipocytes or osteocytes (attached condition), whereas, for the differentiation into chondrocytes (floating condition), the size of sphere is almost the same during the sphere culture. Scale bars: 100 µm.(PDF)Click here for additional data file.Figure S8Teratoma assay. The control testes and the testes injected with LAB-incorporated cell clusters at 14 days after incorporation were extracted, embedded in paraffin and sectioned in 6-µm intervals followed by deparaffinization in xylene and processing through a graded series of alcohol concentrations. The samples were stained with hematoxylin and eosin. Scale bar, 100 µm.(PDF)Click here for additional data file.Figure S9Cell death. To examine cell death, LAB-incorporated cell clusters at 14 days after incorporation were immunostained with rabbit anti-single-stranded DNA antibody (DAKO). After three washes with PBS, the sections (10 µm) were incubated with anti-rabbit IgG antibody conjugated with FITC in the presence of DAPI in the antibody dilutions for 2 h at RT. Scale bar, 100 µm.(PDF)Click here for additional data file.Table S1Comparison between HDF-1,2 and LAB-incorporated cell clusters-1,2.(PDF)Click here for additional data file.Table S2List of 7580 genes that shows the difference by factor of two or more between LAB-incorporated cell clusters-1 vs. HDF-1 and LAB-incorporated cell clusters-2 vs. HDF-2.(PDF)Click here for additional data file.Table S3List of cluster genes that shows the difference by factor of two or more between LAB-incorporated cell clusters-1 vs. HDF-1 and LAB-incorporated cell clusters-2 vs. HDF-2.(PDF)Click here for additional data file.Table S4Selected 37 pluripotency maker genes.(PDF)Click here for additional data file.Table S5List of 50 genes that is decreased by a factor of fifteen or more at least between LAB-incorporated cell clusters-1 vs. HDF-1 and LAB-incorporated cell clusters-2 vs. HDF-2 and has a GO term [development].(PDF)Click here for additional data file.


CRL2LRR-1 promotes germ cell proliferation by counteracting the DNA replication ATL-1 checkpoint pathway. CRL2LRR-1 also participates in the mitotic proliferation/meiotic entry decision, presumably controlling the stability of meiotic promoting factors in the mitotic zone of the germline. Finally, CRL2LRR-1 inhibits the first steps of meiotic prophase by targeting in mitotic germ cells degradation of the HORMA domain-containing protein HTP-3, required for loading synaptonemal complex components onto meiotic chromosomes. Given its widespread evolutionary conservation, CUL-2 may similarly regulate germline development in other organisms as well.The ubiquitin-proteolytic system controls the stability of proteins in space and time. In this study, using a temperature-sensitive mutant allele of the  Maintenance of the germline depends on the presence of a germline stem cell pool with self-renewal potential that produces gametes upon meiotic differentiation. Factors regulating the balance between germline stem cell self-renewal and meiotic differentiation ensure germline homeostasis, whereas disruption of these regulatory mechanisms can lead to sterility or cancer. In this study, we show that the ubiquitin-proteolytic system (UPS), which selectively targets regulatory proteins for proteasomal degradation, controls germline development by acting at three different levels. The UPS promotes germ cell proliferation, regulates the balance between self-renewal and meiotic differentiation, and limits progression through meiotic prophase. In particular, we show that an E3 ubiquitin-Ligase nucleated around the cullin 2 (CUL-2) protein and using the Leucine rich repeat protein LRR-1 as substrate recognition subunit regulates in germ stem cells the stability of HTP-3, which is required for progression through meiotic prophase. These findings identify a previously unknown role for proteolytic regulation in germline development and also explain how the critical balance between germ cell proliferation and meiotic differentiation can be tightly and robustly controlled with multiple, parallel regulatory inputs. The ubiquitin-proteolytic system has emerged as a central mechanism to regulate protein turnover spatially and temporally An enzymatic cascade of three enzymes mediates the attachment of ubiquitin to substrate protein: the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-ligase (E3) Von Hippel Lindau (VHL) complex that regulates the hypoxic response  mutation recapitulates all the cul-2(RNAi) and cul-2(0) phenotypes and also reveals several novel functions in the C. elegans germline. More specifically, we show that besides promoting germ cell proliferation, CUL-2 has at least two other functions in the germline: it participates in the proliferation versus meiotic entry decision and inhibits the first steps of meiotic prophase. Furthermore, we show that the CRL2LRR-1 E3-ligase inhibits meiotic prophase progression at least in part by promoting degradation of HTP-3, a HORMA domain containing protein. HTP-3 is required for progression through meiotic prophase and for the assembly of the synaptonemal complex, the zipper-like structure that tethers homologous chromosomes together.We report here the isolation of a temperature-sensitive mutation in the LRR-1 E3-ligase is essential for germline development in C. elegans: lrr-1(0) and cul-2(0) null mutants are sterile with a small germline lrr-1(0) mutants. While lrr-1(0) animals are sterile, this phenotype is suppressed by inactivation of the DNA replication ATR/ATL-1 checkpoint pathway lrr-1(0) mutants, are sterile at the restrictive temperature of 25°C but recover fertility upon atl-1 depletion by RNAi  mutants, most or209ts mutant animals are sterile at 25°C but recover fertility upon atl-1 inactivation by RNAi  , and we then sequenced the cul-2 gene from or209ts genomic DNA and found a single nucleotide change relative to wild-type in the splicing donor site at the fifth exon-intron boundary (cul-2(or209ts) animals present high levels of embryonic lethality (30%) and a severe reduction in brood size even at the permissive temperature of 15°C, compared to wild-type (cul-2(or209ts) animals present typical cul-2 loss-of-function phenotypes, including the accumulation of abnormally high levels of the TRA-1 protein, presumably causing feminization of the germline  . We nextboundary . This muboundary . Consistild-type . Furthergermline , and of germline , both weor209ts allele, we re-examined CUL-2 function in the germline. The C. elegans germline is spatially organized and contains - from the distal to proximal end -mitotically proliferating stem cells, meiotic germ cells, and gametes (sperm or oocyte). The mitotic zone extends 18–20 germ cell diameters (gcd) along the gonadal axis from the distal end, with the total number of germ cell nuclei exceeding 200 Using the LRR-1 E3-ligase results in a cell cycle arrest cul-2(or209ts) mutants raised from early larval stages (L1) at the restrictive temperature of 25°C. As expected, this phenotype was largely suppressed by inactivation of the ATL-1/DNA replication pathway (cul-2(or209ts) animals, as determined by scoring the distance, in gcd, between the distal end of the germline and the appearance of both nuclear crescents and HIM-3 positive cells (cul-2(or209ts) mutants raised to adulthood at permissive temperature (15°C), and even more reduced in mutants raised at the restrictive temperature (25°C). However, it was difficult to rigorously quantify the phenotype at 25°C because germ cell nuclei were both enlarged and reduced in number (atl-1(RNAi); cul-2(or209ts) mutant hermaphrodites and also observed a smaller mitotic zone in these animals. The size of the mitotic zone was similarly reduced in atl-1(RNAi); lrr-1(0) animals but was unaffected in the control atl-1(RNAi) (zyg-11(b2ts) mutant germlines at 25°C  . Finally at 25°C . Collectcul-2, in addition to regulating mitotic proliferation, might also have a role in preventing meiotic entry, by looking for genetic interactions between cul-2 and genes regulating the proliferation versus meiotic entry decision. GLP-1/Notch signaling controls the decision between self-renewal and entry into the meiotic cell cycle Pumilio and FBF)-domain RNA-binding proteins FBF-1/2 prevent meiotic entry by repressing the translation of meiotic promoting factors and structural components of meiotic chromosomes (cul-2(or209ts) and glp1(bn18), a temperature-sensitive glp-1 mutant in which the Notch signaling pathway is partially defective cul-2(or209ts) and fbf-1(0) or fbf-2(0), and scored meiotic entry in the double versus single mutants at the semi-permissive temperature of 20°C. At this temperature, germ cell proliferation is only modestly affected in cul-2(or209ts) mutants  mutant, as evidenced by the premature meiotic entry in the glp-1(bn18) cul-2(or209ts) double mutants, compared to the single mutants. Likewise, when combined with cul-2(or209ts), both fbf-1 and fbf-2 mutants showed a smaller mitotic zone than single mutants  and molecular markers. More specifically, we monitored the appearance of HIM-3 and SUN-1 Ser8-Pi (P-SUN-1) on meiotic chromosomes  mutants  indicatiEntry into meiosis requires the coordination of a number of events: upon exit from the mitotic zone, germ cells initiate the chromosome dynamics required for meiotic pairing and synapsis. In preparation for this transition, proximal cells in the mitotic zone activate the expression of both regulators of meiotic entry and chromosomal proteins required for synapsis (e.g HIM-3) that are then recruited on meiotic chromosomes. CUL-2 may thus inhibit meiotic entry by controlling the stability of regulators of meiotic entry in the mitotic zone of the germline.cul-2(or209ts) lethality at a semi-permissive temperature (23°C) identified, in addition to ATL-1 and its regulator MUS-101/TopBP1 Interestingly, an RNA interference (RNAi)-based screen  for suppressors of HTP-3 belongs to the family of HORMA (Hop1-Rev1-Mad2) domain-containing proteins LRR-1 in HTP-3 regulation. As expected, the accumulation of HIM-3 on chromosomes in cul-2(or209ts) germ cells depends on HTP-3 because HIM-3 failed to accumulate on chromosomes in htp-3(RNAi); cul-2(or209ts) mutant germlines (htp-3 reduction-of-function suppressed lrr-1(0) mutant sterility. To this end, we took advantage of the htp-3(vc75) allele, which only modestly compromises HTP-3 function lrr-1(tm3543); htp-3(vc75) double mutant animals. Importantly, a significant fraction  of htp-3(vc75); lrr-1(tm3543) double mutant animals were fertile and produced embryos, in contrast to the highly penetrant sterility phenotype observed in lrr-1(tm3543) single mutants (lrr-1(tm3543) sterility by the htp-3(vc75) allele is not merely resulting from an inactive ATL-1 checkpoint pathway in the htp-3(vc75) mutant because the DNA replication checkpoint is fully functional in this mutant  and lrr-1(0) mutant germlines. In control germlines, HTP-3 was expressed at low levels in the most distally located germ cells and at high levels on chromosomes in the most proximal mitotic germ cells (cul-2(or209ts) and lrr-1(tm3543) mutants (atl-1(RNAi); cul-2(or209ts) and atl-1(RNAi); lrr-1(tm3543) germlines, indicating that down-regulation of the DNA replication checkpoint does not affect HTP-3 levels. Importantly, HTP-3 also accumulates in the distal region after inactivation of PBS-5, a proteasome subunit, suggesting that CRL2LRR-1 may target HTP-3 for degradation  because it has been shown recently that CYE-1 depletion forces ectopic proliferative germ cells to load HIM-3 and SUN-1 Ser8-Pi (P-SUN-1) and to enter meiosis GLP-1 promotes the proliferative fate, and the meiotic promoting factors GLD-1, NOS-3, GLD-2 and GLD-3 act downstream of Notch/GLP-1, in two parallel pathways (GLD-1/NOS-3 and GLD-2/GLD-3), to promote entry into meiosis. Simultaneous loss of both pathways causes a defect in meiotic entry that leads to germline overproliferation and prevention of gamete production gld-3 nos-3 germlines upon inactivation of cul-2, as revealed by western blot analysis. We then used immunofluorescence to test whether this accumulation is accompanied by a change in germ cell fate by monitoring the accumulation of the HIM-3 and P-SUN-1 meiotic markers. gld-3 nos-3 germlines inactivated with control dsRNA displayed ectopic proliferating germ cells with no sign of meiotic entry as revealed by the absence of HIM-3 and P-SUN-1 meiotic markers  promotes germ cell proliferation, ii) participates in the proliferation versus meiotic entry decision and iii) inhibits progression through the first step of meiotic prophase by regulating the stability of the axial element HTP-3.Through the isolation and characterization of a unique temperature-sensitive mutant allele of the C. elegans, in particular for genes like CUL-2 with roles in multiple processes. Indeed, ts alleles present numerous advantages: they often only partially reduce gene function even at the fully restrictive temperature, and they allow for modulation of activity through analysis at multiple temperatures.Temperature-sensitive (ts) alleles have been instrumental for discovering the function of essential genes in C. elegans that is highly expressed in the germline and in the early embryo where it has been implicated in numerous processes. Our phenotypic analysis revealed that the cul-2(or209ts) mutant recapitulates all the cul-2 loss of function phenotypes including feminization of the germline (cul-2(or209ts) mutation provides a useful sensitized genetic background for identifying new in vivo functions of CRL2 complexes and, most importantly, for identifying its targets. Finally, other temperature-sensitive mutants affecting the ubiquitin-proteolytic system have been identified in C. eleganscul-2(or209ts) is to our knowledge the first conditional mutation reported for a metazoan cullin.CUL-2 is an essential gene in germline , defectsgermline , and defcul-2(or209ts) allele, we confirmed our previous observations indicating that loss of the CRL2LRR-1 E3-ligase causes hyperactivation of the ATL-1/DNA replication checkpoint in germ cells, resulting in a cell cycle arrest and adult sterility cul-2(or209ts) mutant at 25°C, a phenotype that was partially suppressed by RNAi-mediated inactivation of atl-1 (mus-101 and atl-1 function by RNAi increased the hatching rate of the cul-2(or209ts) mutant (LRR-1 function triggers activation of the ATL-1/DNA replication checkpoint pathway.Using the of atl-1 . Further) mutant , furthercul-2(or209ts) and lrr-1(0) mutants? We believe that one function of the CRL2LRR-1 complex is to regulate DNA replication integrity, both in germ cells and in early embryos lrr-1(0)cul-2(or209ts) mutants (this study). In addition, our observations suggest that the inappropriate accumulation of structural components of meiotic chromosomes in lrr-1(0) mutants may also contribute to the activation of the ATL-1 checkpoint pathway (see below).Why is the ATL-1 checkpoint pathway hyperactivated in C. elegans germline, GLP-1/Notch signaling controls the decision between self-renewal and entry into the meiotic cell cycle cul-2ts mutant enhanced the premature meiotic entry phenotype of the glp-1(bn18) mutant and when combined with the cul-2ts mutant, both fbf-1 and fbf-2 mutants showed a smaller mitotic zone than single mutants , suggesting that CUL-2 might not be involved in GLD-1 degradation. Nevertheless, we cannot exclude the possibility that a small fraction of GLD-1 accumulates upon inactivation of cul-2 and thereby contributes to the cul-2 phenotype.Alternatively, CUL-2 might act with CYE-1/CDK-2 to control the stability of meiotic promoting factors in the mitotic zone of the germline. Consistent with this hypothesis, substrate phosphorylation often is a pre-requisite for recognition by an E3 ligase LRR-1 negatively regulates progression through meiotic prophase by controlling the stability of HTP-3  allele or with RNAi, suppressed lrr-1(tm3543) sterility and increased the hatching rate of the cul-2(or209ts) mutant, respectively. In addition, reducing syp-1 function also increased the hatching rate of the cul-2(or209ts) mutant. These results suggest that premature accumulation of HTP-3 on chromosomes allows for inappropriate HIM-3 and subsequently SYP-1 binding. This inappropriate assembly of SC components on chromosomes likely contributes to DNA replication checkpoint activation. However, although SC components appear to be expressed and loaded inappropriately on chromosomes upon inactivation of the CRL2LRR-1 E3-Ligase, germ cells do not appear to enter into meiosis because we failed to detect SUN-1 Ser8-Pi on chromosomes of ectopic proliferating germ cells upon inactivation of cul-2 or lrr-1. This observation indicates that HTP-3 accumulation is not sufficient to drive progression through meiotic prophase and that CUL-2 acts independently of HTP-3 to influence to proliferation versus meiotic entry decision, presumably by controlling the stability of meiotic regulator(s) in the mitotic zone of the germline II/mIn1[mIs14 dpy-10(e128)]IIcul-2(or209ts)IIIhtp-3(vc75)Ihtp-3(vc75)I; lrr-1(tm3543)II/mIn1[mIs14 dpy-10(e128)]II (this study); glp-1(bn18)atl-1(tm853)IVgld-3(q730) nos-3(q650)II/mIn1[mIs14 dpy-10(e128)]IIgld-3(q730) nos-3(q650)II/mIn1[mIs14 dpy-10(e128)]II; cul-2(or209ts)III (this study), fbf-1(ok91)IIglp-1(bn18) cul-2(or209ts)III (this study); fbf-1(ok91)II; cul-2(or209ts) III (this study); fbf-2(q738)IIfbf-2(q738)II; cul-2(or209ts)III (this study).or209 worms were mated to the Hawaiian CB4856 C. elegans wild isolate, and recombinant F2 lines homozygous for the or209 mutation were isolated based on embryonic lethality at the restrictive temperature. Several SNPs located between −27.2 cM and 21.25 cM along the third chromosome were amplified by PCR from these recombinants, essentially as described or209 mutation between +18.52 cM and the right end of chromosome III. This region contains cul-2, and we showed that all embryos from or209/cul-2(ek1) trans-heterozygotes  The results are presented as means ± S.E.M. In all graphs, data were compared by a Mann-Whitney test (two-tailed p) or Student test . All cal3).L4 animals were fed on NGM plates containing 10 mM final HU prior to analysis. Germlines were then dissected and stained with DAPI. Quantification of HU-induced cell cycle arrest was performed by counting the number of nuclei in a defined volume , and incubated overnight at 4°C with primary antibodies diluted in PBT. Working dilutions for the primary antibodies were 1∶1000 for rabbit anti-HIM-3 (M. Zetka), 1∶100 for rabbit anti-HTP-3 (M. Zetka) and 1∶1000 anti-SUN-1 Ser8-Pi (V. Jantsch). Slides were later incubated for 30 min at room temperature with secondary antibodies coupled to the Alexa 488 and 568 fluorophore . Next, germlines were mounted in Vectashield Mounting Medium with DAPI (Vector). Fixed germlines were imaged using either a TCS SP5 confocal microscope (Leica) or a LSM 710 confocal microscope (Zeiss) with 40× objectives. Confocal images correspond to the projection of confocal Z-stacks spanning maximum 5 µm. Captured images were processed using ImageJ and Adobe Photoshop.Germlines were dissected in PBS followed by freeze-crack, immersion in cold MeOH (−20°C) for 1 min, and fixation in 1× PBS, 0.08 M HEPES (pH 6.9), 1.6 mM MgSOFor whole-worm images, worms were immobilised with 20 mM levamisole and mounted on 2% agarose pads. Images were then acquired using an Axiovert 200 inverted microscope equipped with DIC optics.Standard procedures were used for SDS–PAGE and western blotting. The following antibodies were used in this study: primary antibodies were directed against CUL-2 E. coli and purified as described LRR-1/ELC-1/ELB-1 trimeric complexes were expressed in Total worm extracts were prepared by cryolysis, as previously described Figure S1cul-2 gene. A- Flow chart of the approach used to screen temperature-sensitive mutants affecting the function of the CRL2LRR-1 complex. The basic idea is to screen for mutants that behave like lrr-1(0) mutant animals. lrr-1(0) animals are sterile with a small germline but this phenotype is fully suppressed by inactivation of the ATL-1/DNA replication checkpoint pathway. Furthermore, in the early embryo, depletion of lrr-1 by RNAi causes a specific delay in the division of the P1 germline blastomere (Div phenotype for cell division defective). Therefore, we screened a collection of temperature-sensitive div mutants that are sterile at 25°C but recover fertility upon inactivation of the ATL-1 checkpoint pathway. The div mutants were isolated essentially as described. div(or) mutant L1 larvae were fed with control or atl-1 dsRNA at restrictive temperature (25°C) until adulthood and the percentage of fertile animals was determined by DIC microscopy. B- Graph showing the percentage of fertile div(or) mutants after control (dark bars) or atl-1(RNAi) (grey bars). C-Mapping of div(or209ts) mutation to the end of chromosome III is shown. The ratio of or209 N2 DNA (blue bars) to “Hawaiian” DNA (orange bars) measured at various single nucleotide polymorphisms ) are presented on the graph; cul-2 localised at 21.36 cM. D- Embryonic lethality and brood size of cul-2(or209ts) mutant animals. Graphs show the embryonic viability (left panel) and brood size (right panel) of WT (dark bars) and cul-2(or209ts) (grey bars) animals at permissive temperature (15°C).Identification of a temperature-sensitive allele in the (TIF)Click here for additional data file.Figure S2cul-2(or209ts) embryos. Fluorescent micrographs of cul-2(or209ts) embryos expressing GFP::PIE-1 are shown. Note the mislocalisation of GFP::PIE-1 to somatic blastomeres in cul-2(or209ts) embryos produced at 25°C (right panels).Mislocalisation of PIE-1 in (TIF)Click here for additional data file.Figure S3lrr-1(tm3543); htp-3(vc75) double mutants. A- Lineage diagram on the right, vertical bars indicate time, horizontal bars indicate cell division, anterior daughters are positioned to the left, and posterior daughters to the right. B- The elapsed time, in seconds (s), between AB and P1 cytokinesis was determined and plotted. Note that the ATL-1 checkpoint pathway is hyperactivated in lrr-1(tm3543); htp-3(vc75) double mutant embryos as revealed by a severe delay in the division of the posterior P1 blastomere in two-cell stage embryos.The ATL-1/DNA replication checkpoint pathway is hyperactivated in (TIF)Click here for additional data file.Table S1div alleles.List of (TIF)Click here for additional data file.


HAART has greatly modified the course of HIV-1 infection; however, its impact seems to be less favourable on lymphoproliferative disorders associated with Epstein-Barr virus (EBV) than on other AIDS-defining illnesses. The aim of this study was to estimate the relationship between EBV levels and other viro-immunological parameters in HIV-1 infected subjects in the HAART era.164 HIV-1 infected patients (pts) who attended the Infectious Diseases Unit of Rovigo Hospital, from July 2007 to December 2009 were included in this study. 28% of patients had HBV and/or HCV coinfections. HIV-1 RNA in plasma was quantified by COBAS Taqman HIV-1 test. HIV-1 DNA and EBV-DNA in peripheral blood mononuclear cells (PBMC) were determined by real-time PCR. Lipopolysaccharide (LPS), a marker of microbial translocation, was determined in plasma samples using a chromogenic assay (Limolus Amebocyte Lysate). B-cell activation was analyzed by flow cytometry using monoclonal antibodies CD19PerCP, CD86APC, and CD69PE.The median (IQR) EBV-DNA load was 41(1-151) copies/105 PBMC. 48% of pts had CD4 >500 cells/µl and 27% had undetectable HIV viral load. The EBV-DNA level was significantly higher in pts with CD4 below 500 cells/µl than in those with CD4 >500cells/µl  and in pts with detectable HIV-1 RNA than in those with undetectable viremia . Levels of EBV-DNA were higher in the group of pts with CD4 cell counts >500 cells/µl and high HIV-1 viremia (>1000 copies/ml) than in pts with low viremia, regardless of the immunological status . Furthermore, pts with high EBV loads (up to 75th percentile) had higher levels of HIV-1 DNA [40(1-132) vs 10(1-76) HIV-DNA copies/105; p= 0.050) and higher levels of LPS [130(88-244) vs 98(81-134) pg/ml; p=0.024) than pts with low EBV loads. B-cell activation in pts with high EBV loads was confirmed by immunophenotyping; two of these pts developed a B-cell lymphoma.These findings suggest that HIV-1viremia, other coinfections, and immune activation play an important role in the B-cell stimulation and expansion of EBV-infected cells. Persistent HIV-1 viremia, despite immunereconstitution, may represent a risk factor for the onset of EBV-related cancers.


Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each  Caenorhabditis elegans genome, a conserved sequence motif lies upstream of many piRNA loci and appears to regulate their expression. We combined computational and experimental approaches to investigate the role of this motif in the expression of C. elegans piRNAs. We discovered that >70% of piRNAs are differentially enriched in male versus female germline, and these male and female piRNAs show different upstream motifs. Using a transgenic system for expressing synthetic piRNAs in vivo, we demonstrate that variation of a single nucleotide within this motif influences piRNA germline enrichment. We further show that the conserved motif is capable of driving piRNA expression in genomic isolation. Accordingly, the genomic distribution of these motifs determines which sequences are expressed as piRNAs in C. elegans. Our results suggest that each C. elegans piRNA represents an independent transcript whose sequence, abundance, and germline enrichment are encoded by a variant upstream motif, defining a novel modality for expression of piRNAs.Across the animal kingdom, Piwi-interacting small RNAs (piRNAs) protect genome integrity and promote fertility. While the functions of piRNAs are well-characterized, far less is known about how they are generated and how their expression is regulated. In the  In fly and mouse, primary piRNAs appear to be processed from long, single-stranded RNA precursors Caenorhabditis elegans. They are terminally methylated C. elegans piRNAs exhibit some unusual features. While the vast majority of 21U RNAs map to two large genomic clusters on chromosome IV, the loci do not exhibit prominent strand biases 21U RNAs, a class of germline-enriched small RNAs, represent the piRNAs of C. elegans. Combining computational and transgenic approaches, we find that the conserved core motif defines the piRNA transcriptional cassette, specifying expression of 21U RNAs from genomic thymidines situated at an optimal distance downstream to determine which genomic sequences are expressed as C. elegans piRNAs. Core motifs also encode information dictating germline-specific expression of 21U RNAs. We show that more than 70% of C. elegans piRNAs are preferentially enriched in male or female germline. Unexpectedly, this germline enrichment appears to be enforced by a single nucleotide position within the core motif. We demonstrate autonomous expression of synthetic 21U RNAs from multiple minimal transgenic cassettes consisting only of the 8 nt core motif, the ∼40 nt intervening genomic spacer, the 21U RNA sequence, and ∼50–100 nt of flanking genomic context. Finally, we use single-copy transgenes integrated in genomic isolation to show that the clustered organization of endogenous piRNA loci is entirely dispensable for robust piRNA expression. Together, our results suggest that each 21U RNA locus encodes all of the information necessary for driving independent, autonomous transcription from more than 15,000 unique piRNA loci in C. elegans.Here, we demonstrate that piRNAs are expressed autonomously in fem-1(hc17) adult female versus him-8(e1489) or fog-2(q71) adult male animals shows segregation of 21U RNAs according to germline enrichment classification  . The trend is corroborated by Taqman analysis showing depletion of male 21U RNAs and enrichment of female 21U RNAs in embryo  . Furthern embryo –S2E. Theprg-1(n4357) deep sequencing datasets, as these are likely not 21U RNA-dependent, we identified 11,377 (72.3%) unique 22G RNAs that are likely male 21U RNA-dependent and 3,855 (24.5%) unique 22G RNAs that are likely female 21U RNA-dependent . We then compared the 5,956 male and 1,387 female 21U RNA targets identified in young adult 2 test, p = 7.7e−13)  .2 test, p = 0.044), but neither enriched nor depleted for oogenesis transcripts  , as expected, but female 21U RNA targets are significantly enriched for oogenesis transcripts . These differences between male and female 21U RNA targeting suggest that the evolutionary pressures acting on male and female 21U RNA sequences may differ  . Curious = 0.76) . Their tffer See .GTTTCACT, is separated from the 21U RNA locus by an A/T-rich spacer of ∼35 to 42 nt GTTTC of the core motif, we found that 6,615 of 7,677 (88%) male 21U RNAs show a canonical, GTTTC-containing core motif, compared to only 1,119 of 2,171 (54%) female 21U RNAs. While the length of the A/T-rich spacer does not differ between male and female 21U RNAs  female 21U RNAs   . To examC. briggsae unc-119 gene  germline-deficient mutant  and is highly expressed in him-8(e1489) male adult; in contrast, expression of endogenous ♀21UR-2502, with an ATGTTTCA core motif, peaks after the spermatogenesis-to-oogenesis transition in adulthood (∼72 h) and is highly expressed in fem-1(hc17) female adult (CTGTTTCA to ATGTTTCA (♂C>A1258 transgene) or ATGTTTCA to CTGTTTCA (♀A>C2502) disrupts these germline-specific expression patterns. Whereas 21UR-synth expression from ♂Tg1258 plummets after spermatogenesis, loss of the core motif 5′ cytidine in the ♂C>A1258 transgenic strain results in sustained 21UR-synth expression through oogenesis; the ♂C>A1258 transgene also preferentially expresses 21UR-synth in fem-1(hc17) female (fem-1(hc17) female, indicating that other elements contribute to female 21U RNA expression patterns. This is consistent with our finding that female 21U RNA core motifs show no bias at the 5′ nucleotide, and indeed ∼21% of female 21U RNA core motifs show a 5′ cytidine (prg-1 (We then used our transgenic system to test whether variation at the core motif 5′ position affects germline expression of 21UR-synth . Endogenle adult . Accordi) female . Thus, m) female . These rcytidine . As expee  or guanosine (21U>G transgenes), such that the transgenes encode 21[U>A]R-synth or 21[U>G]R-synth, respectively . Therefore, the presence of multiple thymidines within the optimal genomic window downstream of a core motif might result in expression of multiple, overlapping 21U RNAs. Indeed, many GTTTC-containing core motifs . Additionally, we observed significantly greater thymidine richness in the optimal genomic windows 35–42 nt downstream of shared GTTTC-containing motifs versus non-shared   . Therefo2 test, p = 9.6e−28). We note that this paucity of mixed male∶female 21U RNA miniclusters likely contributes to the low number of 22G RNAs that can be attributed to both male and female 21U RNAs . Random assortment of these 21U RNAs would predict 66% male∶male, 4% female∶female, and 31% male∶female pairs; however, we observed 73% male∶male, 12% female∶female, and only 15% male∶female pairs. Thus, 85% of pairs showed matching enrichment classification , a signi21U RNAs .C. elegans genome and the presence of the conserved upstream motif have generated speculation that 21U RNAs represent autonomously transcribed units prg-1 dependence, rde-4 independence, and germline enrichment as endogenous 21U RNAs  than on chromosome IV outside these regions (0.4 occ/kb) or on other chromosomes (0.2 occ/kb). Furthermore, 21U RNAs encoded on chromosome IV are detected at much higher abundance (mean abundance: 148 RPM) than those encoded on other chromosomes (1 RPM) . These observations suggest the possibility of a positional requirement for expression of 21U RNA loci: a privileged genomic environment might contribute to the expression of 21U RNAs. To investigate the significance of 21U RNA genomic organization, we carried out rough mapping of the genomic insertion sites of several of the high-copy transgenic arrays. None of the integration loci mapped to chromosome IV ], xkIs12[♀Scram2502 cb-unc-119(+)], xkIs14[♂21U>A1258 cb-unc-119(+)], xkIs15[♂21U>G1258 cb-unc-119(+)], xkIs16[♂Min1258 cb-unc-119(+)], xkIs17[♀21U>A2502 cb-unc-119(+)], xkIs18[♀21U>G2502 cb-unc-119(+)], xkIs19[♀Min2502 cb-unc-119(+)], xkIs20[♂Min1415 cb-unc-119(+)], xkIs21[♂MinC>A1415 cb-unc-119(+)], xkIs22[Tg1415-Tg2109 cb-unc-119(+)], xkIs23[Scram1415-Tg2109 cb-unc-119(+)]; LGX: xkIs10[♀A>C2502 cb-unc-119(+)]; LGI: glp-4(bn2), prg-1(tm872), xkIs5[♀Tg2502 cb-unc-119(+)]; LGII: xkSi30 [♀Min2502 cb-unc-119(+)], xkIs6[♂C>A1258 cb-unc-119(+)]; LGIII: rde-4(ne301), henn-1(tm4477); LGIV: xkSi3[♂Tg1258 cb-unc-119(+)], xkSi13[♀Tg2502 cb-unc-119(+)], xkSi17[♂C>A1258 cb-unc-119(+)], xkSi20[♀A>C2502 cb-unc-119(+)], xkSi23[♂Scram1258 cb-unc-119(+)], xkSi28[♀Scram2502 cb-unc-119(+)], fem-1(hc17), him-8(e1489); LGV: fog-2(q71), xkIs1[♂Tg1258 cb-unc-119(+)]. Transgenic allele details and corresponding strain names are shown in C. elegans samples were generated as previously described MASGSGRGRGRGSGSNNS (C)) conjugated to keyhole limpet hemocyanin (KLH) carrier protein. Antisera were affinity purified using Affi-Gel 10 gel (Bio-Rad). PRG-1 was purified from synchronized gravid animals using this anti-PRG-1 rabbit polyclonal antibody. For each IP, 10 µg of anti-PRG-1 antibody was cross-linked to Dynabeads Protein A (Invitrogen) and incubated with lysate prepared from 0.3 ml of frozen worms at 4°C for 1 hr. Beads were washed 4× with RIP wash buffer . After final wash, beads were split into equal volumes for RNA extraction and western blot procedure. For western blot analysis: 30 ul of 1× Tris-glycine SDS sample buffer (Invitrogen) without DTT was added directly to beads and incubated at 50°C for 10 min. 0.1 M DTT was then added to samples and boiled for 5 min before loading on gel. Proteins immobilized on Immobilon-FL transfer membrane (Millipore) were probed with anti-PRG-1 rabbit polyclonal antibody or anti-gamma-tubulin rabbit polyclonal antibody (LL-17) (Sigma) . Peroxidase-AffiniPure goat anti-rabbit IgG secondary antibody was used at 1∶10,000 (Jackson ImmunoResearch Laboratories) for detection using Pierce ECL Western Blotting Substrate (Thermo Scientific). For RNA extraction: 1 ml of TRI-Reagent (Ambion) was directly added to beads and incubated at room temperature for 5 min. RNAs were precipitated in isopropanol for 1 hr at −30°C followed by three washes with 70% ethanol.RNA isolation, beta-elimination, northern blot analysis, Taqman RT-qPCR, and mRNA quantitation were performed as previously described Cbr-unc-119 qPCR data were normalized to act-1 mRNA levels. The sequence of 21UR-synth is 5′ TGATATGCGATGTAGTAGACT 3′. The sequence of 21UR-synthB is 5′ TTAGTCGTATGTGACGCTGCC 3′. Full small RNA sequences were submitted to Applied Biosystems for design of Taqman assays. Northern blot probe sequences used for this study: miR-1 5′ TACATACTTCTTTACATTCCA /3StarFire/ 3′; ♀21UR-2502 5′ CAGCAGTCTACTACAATTTCA /3StarFire/ 3′; 21UR-synth 5′ AGTCTACTACATCGCATATCA /3StarFire/ 3′. RT-qPCR primer sequences used for this study are as follows: act-1 F 5′ CCAGGAATTGCTGATCGTATGCAGAA 3′, R 5′ TGGAGAGGGAAGCGAGGATAGA 3′; Cbr-unc-119 F 5′ AACGACGTTTTAGCACTTCCG 3′, R 5′ GGATTTGGAACTTGGTGAACTCG 3′.Small RNA quantitation was performed as previously described ♂Min1258 transgene. To generate the base of the 2502 transgene, sequence spanning genomic coordinates IV:15395699–15397722 was used; IV:15396667–15396886 was used for the ♀Min2502 transgene. To generate the base of the Min1415 transgene, sequence spanning genomic coordinates IV:16564187–16564395 was used. To generate the base of the Tg1415–Tg2109 transgene, sequence spanning genomic coordinates IV:16564133–16564395 was used; the Tg1415–Tg2109 and Scram1415-Tg2109 transgenes carry a 13 nt deletion downstream of both 21U RNA loci. Coordinates were taken from the C. elegans genome WS220. The mutations described in To generate the base of the 1258 transgene, sequence spanning genomic coordinates IV:14390835–14393692 was used; IV:14392513–14392673 was used for the Raw data files from 24 small RNA sequencing experiments C. elegans genome version WS220 using Bowtie C. elegans genome version WS220 using Bowtie. Perfect, full-length alignments for 15,093 of these sequences were considered valid 21U RNA coordinates. Reads mapping entirely within these coordinates were annotated to 21U RNAs.Reads were aligned to the reference prg-1(tm872) young adult library compared to an N2 young adult library Germline enrichment classifications of 21U RNAs were generated based on read counts in 17 germline libraries: 14 male germline libraries prepared from isolated spermatogenic cells, isolated spermatids, or whole adult males; and 3 female germline libraries prepared from purified oocytes or whole adult hermaphrodites defective in sperm production . 1,198 2To approximate the number of 21U RNAs falsely classified as male or female germline-enriched by our method, we performed Enrichment Score calculations on randomly generated count data modeled from an N2 young adult library 26G RNA annotations were taken from Han et al., 2009 prg-1(n4357)) was obtained from Bagijn et al. prg-1(n4357)) were downloaded from GEO 21U RNA target and 22G RNA information for young adult animals  defined as the start of 5′ UTRs annotated in the Ensembl66 database fragments per kilobase per million mapped reads (FPKM) using biological replicates from a transcriptomic sequencing experiment C. elegans genome version WS220 using TopHat Young adult TSS expression was calculated as Published nucleosome occupancy data Pol II ChIP-seq data from young adult worms were downloaded from the modEncode repository. Pol II signal to input ratios were averaged for each nucleotide separately for male and female 21U RNAs on chromosome IV.Dataset S1List of 21U RNAs analyzed in this study, their Enrichment scores, their germline enrichment classifications based on Enrichment scores (Classification by score), and final germline enrichment classifications after removal of 21U RNAs whose average fold abundance in the enriched germline was not higher than the non-enriched germline (Adj. for avg. fold abundance).(XLSX)Click here for additional data file.Figure S1Computational identification of male and female germline-enriched 21U RNAs. (A) Enrichment Score calculations performed on 17 small RNA sequencing libraries classify a majority of 21U RNAs as male (blue) or female (red) germline-enriched. Non-enriched (NE) 21U RNAs, grey. Numbers indicate percent of 13,711 21U RNAs analyzed. (B) Enrichment Score calculations performed on control data classify <1% of 21U RNAs as male or female germline-enriched indicating a 1% false discovery rate. Numbers indicate percent of 11,458 21U RNAs analyzed. (C) Male 21U RNAs are more abundant in male libraries. Average relative abundance of each male 21U RNA was calculated between each of the 23 male∶female library comparisons. (D) Female 21U RNAs are more abundant in female libraries. Average relative abundance of each male 21U RNA was calculated between each of the 23 female∶male library comparisons. (E) Non-enriched 21U RNAs are equally abundant in male and female libraries.(PDF)Click here for additional data file.Figure S2him-8(e1489) (C) and fog-2(q71) (D) male animals and N2 embryos. Error bars represent ±1 SD from two biological replicates. (E) Taqman RT-qPCR analysis corroborates female 21U RNA enrichment in embryo. Expression of representative female 21U RNAs was assayed by Taqman in fem-1(hc17) female animals and N2 embryos. (F) Male germline-enriched 26G RNAs are generally absent in embryo. Average relative abundance of each male 26G RNA was calculated between each of 4 male and 4 mixed stage embryo libraries. (G) Female germline-enriched 26G RNAs are robustly expressed in embryo. Average relative abundance of each female 26G RNA was calculated between each of 1 female and 4 mixed stage embryo libraries.Female 21U RNAs are preferentially abundant in embryo. (A) Relative male 21U RNA abundance is decreased in embryo. Average relative abundance of each male 21U RNA was calculated between each of 5 male and 4 mixed stage embryo libraries. Dotted line indicates equal male and embryo abundance. Pie chart depicts proportion of male 21U RNAs with reads in at least one embryo library (dark blue). (B) A population of female 21U RNAs shows increased abundance in embryo. Average relative abundance of each female 21U RNA was calculated between each of 1 female and 4 mixed stage embryo libraries. Pie chart depicts proportion of female 21U RNAs with reads in at least one embryo library (dark red).  Taqman RT-qPCR analysis corroborates male 21U RNA depletion in embryo. Expression of representative male 21U RNAs was assayed by Taqman in (PDF)Click here for additional data file.Figure S321U RNAs target significantly non-overlapping sets of genes. (A) 22G RNAs are almost exclusively derived from either male or female 21U RNAs, but not both. The number of unique 22G RNAs derived from both male and female 21U RNAs is significantly less than expected if 22G RNAs are selected at random . (B) Male and female 21U RNAs target significantly fewer overlapping genes compared to selecting random sets of genes . (C) 5,956 genes targeted by male 21U RNAs in young adult (YAd) animals are depleted of spermatogenesis genes compared to a random set of 5,956 genes. (D) 1,387 genes targeted by female 21U RNAs in gravid adult (GA) animals are enriched for oogenesis genes compared to a random set of 1,387 genes.(PDF)Click here for additional data file.Figure S4Cbr-unc-119 mRNA in adult animals were assayed by RT-qPCR for all transgenes and normalized to act-1 mRNA levels. (B) Expression of transgenic 21UR-synth does not affect expression of endogenous 21U RNA counterparts. Endogenous ♂21UR-1258 and ♀21UR-2502 levels were assayed by Taqman RT-qPCR and normalized to microRNA miR-1 levels in all samples.Transgenic array expression varies across transgenes. (A) Levels of (PDF)Click here for additional data file.Figure S521U RNAs are specifically immunoprecipitated with PRG-1 complexes. (A) anti-PRG-1 antibody does not immunprecipitate microRNA miR-1. (B) 21UR-synth expression does not interfere with association of endogenous 21U RNAs with PRG-1.(PDF)Click here for additional data file.Figure S621U RNA expression requires a 5′ genomic thymidine. (A) Schematic of transgenes encoding 21UR-synth with different 5′ nt. (B–C) Mutation of the 5′ genomic thymidine disrupts expression of 21UR-synth by northern blot (B) and Taqman assay (C). WT and ♀Tg2502 lanes in (B) are repeated from (PDF)Click here for additional data file.Figure S7st quartile) and the bottom 25% (4th quartile) separately. 21U RNAs on all chromosomes are shown. (D) Same as (E) but showing top and bottom 25% of TSS by transcript abundance.RNA polymerase II occupancy at 21U RNA loci is below background level. (A) Average Pol II occupancy in a young adult library of 21U RNA loci expressing 21U RNAs with at least 5 RPM (red), transcriptional start sites (TSS) expressing transcripts with at least 5 FPKM (green), and randomized intergenic regions (yellow). Only regions on ChrIV were assayed (B) Pol II occupancy as described in (A) but independently scaled for each transcript type and plotted with average nucleosome occupancy (black line). Grey error bands: SEM. (C) Average Pol II occupancy of 21U RNA loci as (B) but showing the top 25% 21U RNAs by abundance (1(PDF)Click here for additional data file.Figure S8Model of 21U RNA expression.(PDF)Click here for additional data file.Table S1Descriptions of small RNA sequencing libraries used in this study. GEO Accessions for datasets and libraries used are listed. Libraries generated using 5′-monophosphate-dependent (Dep) or -independent (Indep) RNA extraction protocols are indicated along with how the library was used in this study (“Use” column).(PDF)Click here for additional data file.Table S2t-test p-values for all abundance comparisons between 21U RNAs with different core motifs. Highlighted are p-values <0.01. Identity of the 5′ nt corresponding to higher 21U RNA abundance is indicated below each significant p-value. All t-tests are two-tailed. Comparisons of abundances in 5′-monophosphate-dependent and -independent libraries were performed separately.Welch's (PDF)Click here for additional data file.


Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644) mutants. SLX-1 shows a HIM-18/SLX-4–dependent endonuclease activity toward replication forks, 5′-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs) along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs.Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the  Caenorhabditis elegans, a single off-centered crossover is formed on the chromosome arms between every pair of homologous chromosomes. Crossover formation at the central region of the chromosomes is suppressed by unknown mechanisms. By using high-resolution 3-D microscopy, we found that, while crossover distribution is biased to the arm regions along the chromosomes, DNA double-strand breaks (DSBs), which initiate the homologous recombination repair process, are evenly distributed along the chromosomes. These results suggest the existence of mechanisms that inhibit crossover formation after induction of DSBs at the central region of the chromosomes. In this study, our findings lead us to hypothesize that SLX-1, a structure-specific endonuclease, inhibits crossover formation at the central region of the chromosomes, probably via its resolution activity of the Holliday junctions, which are four-stranded recombination intermediates, to produce noncrossover products.Crossover formation between homologous chromosomes is important for generating genetic diversity in subsequent generations, as well as for promoting accurate chromosome segregation during meiosis, which is a specialized cell division program that results in the formation of haploid gametes (sperm and eggs) from diploid parental germ cells. In the nematode  Escherichia coli, shows endonuclease activity towards Y forks, 5′-flaps, RFs and HJs E. coli, cleaves Y forks, 3′-flaps, 5′-flaps, RFs, stem loops and HJs Genomic DNA is subjected to a variety of endogenous and exogenous sources of damage. To repair this DNA damage, the structure-specific endonucleases function to cleave branched DNA structures such as Y forks, 5′-flaps, 3′-flaps, stem loops, bubbles, replication forks (RFs) and Holliday junctions (HJs). SLX-1, a structure-specific nuclease that is highly conserved in eukaryotes, harbors both GIY-YIG-type endonuclease and PHD-type zinc finger domains. Furthermore, SLX1 requires the regulatory subunit SLX4 to perform its nuclease activities in both yeast and humans slx1 was first identified in a synthetic-lethal screen for genes required for the viability of cells lacking Sgs1, the budding yeast RecQ helicase slx1 deletion (slx1Δ) mutants do not exhibit lethality, DNA damage sensitivity or sterility in either budding or fission yeast SLX4 exhibit symptoms of Fanconi anemia, a syndrome characterized by chromosomal instability in humans XPF in a mutually exclusive manner C. elegans, interacts with SLX-1 and XPF-1 in vivo is also unclear.Caenorhabditis elegans SLX-1 cleaves branched DNA substrates in a HIM-18/SLX-4-dependent manner in vitro. Furthermore, slx-1(tm2644) mutants, which encode for a catalytically inactive (nuclease negative) protein, show accumulation of recombination intermediates in both mitotic and meiotic cells as well as increased sensitivity to DNA damage inducing agents. These results suggest that SLX-1 is required for DNA repair by processing repair intermediates through its nuclease activity. However, while double Holliday junction resolution is required for crossover formation during meiosis, meiotic crossover frequencies were not reduced in slx-1(tm2644) mutants, and instead, crossover distribution was altered compared to wild type. Therefore, although SLX-1 is not an essential nuclease for crossover formation via double Holliday junction resolution between homologous chromosomes during meiotic recombination, our studies reveal that SLX-1 plays a role in regulating crossover distribution. This regulation is not mediated through changes in either the levels or distribution of DNA double-strand breaks (DSBs) along chromosomes. Instead, we propose a model in which SLX-1 regulates meiotic crossover distribution such that they occur at the terminal thirds of chromosomes by promoting DSB repair via a noncrossover pathway along the mid-section of chromosomes.Here we show that C. elegans SLX-1 interacts with HIM-18 in a yeast two-hybrid assay We previously showed that To examine the roles of HIM-18 and SLX-1 during recombination, we assessed if the HIM-18/SLX-1 complex displayed endonucleolytic activity towards synthetic DNA substrates. HA-HIM-18-CCD  and Myc-SLX-1 were co-expressed in 293T cells and immuno-purified with an α-HA antibody (the CCD domain of HIM-18 was used since it expressed at a higher level than full-length HIM-18). The HA-HIM-18/Myc-SLX-1 complex was incubated with either a radiolabeled replication fork (RF) or a Holliday Junction (HJ) substrate, and the reaction products were separated by native gel electrophoresis and visualized by autoradiography. HA-HIM-18/Myc-SLX-1 exhibited endonucleolytic activity against both RFs and HJs at a level that was comparable to the human HA-SLX4-ΔN complexes purified from 293T cells  [5]. UndC. elegans protein complex  mutant, obtained from the Japanese National Bioresource Project, carries a 205 bp deletion encompassing parts of intron 4 and exon 5 which removes the splice acceptor site of the downstream exon 4  mutants  mutants and stems from the only splice variant still carrying the nuclease domain, does not bind to HIM-18 and lacks a nuclease activity (slx-1(tm2644) mutants are loss-of-function for SLX-1's nuclease activity.The m exon 4 . RT-PCR  mutants . Sequenc mutants . SLX-1 h mutants . 42% of  mutants , whereas mutants . Both ex mutants . Howeveractivity . Therefoslx-1 plays a role in either mitotic development or meiosis, and examine the genetic interactions between slx-1 and other genes implicated in processing recombination intermediates during these cell division programs, we measured the brood size, embryonic lethality, larval arrest and the incidence of males observed among slx-1, him-6/BLM, xpf-1 and gen-1 mutant offspring (slx-1 mutants showed a 32% reduction in brood size (P<0.0001), a 4.6-fold increase in embryonic lethality (P = 0.0006), and a 2-fold increase in larval arrest (P = 0.00197) compared to wild type, supporting a role for slx-1 in mitotic repair. Moreover, a 1.2-fold increase in larval arrest (P = 0.0417) was observed in slx-1;him-18 double mutants compared to him-18 single mutants. This marginally lower significance cutoff most likely indicates that SLX-1 is dependent on HIM-18 during larval development. However, we cannot rule out the possibility that SLX-1's function is not completely HIM-18-dependent during this developmental process. Next, we investigated the genetic interaction of SLX-1 with HIM-6, a C. elegans homolog of yeast Sgs1 and the human Bloom syndrome helicase, which is required for double Holliday junction dissolution slx-1;him-6 double mutants showed synthetic lethality compared to either single mutant (P<0.0001). These results suggest that SLX-1 and HIM-6 function in parallel or alternate pathways, similar to budding yeast slx-1 and the structure-specific endonucleases xpf-1 and gen-1, which are a component of the HIM-18 complex and a Holliday junction resolving enzyme, respectively slx-1;xpf-1 double mutants showed a 2.4-fold increase (P<0.0001) in embryonic lethality and a 3.6-fold increase in larval arrest (P = 0.0029) compared with xpf-1 single mutants. These results suggest that SLX-1 and XPF-1 may have overlapping roles during mitotic development. Interestingly, slx-1;xpf-1;him-18 triple mutants exhibited similar phenotypes to xpf-1;him-18 double mutants, supporting our observation that the nuclease activity of SLX-1 is dependent on HIM-18 and therefore an slx-1 mutation causes no obvious plate phenotypes in an xpf-1;him-18 background. Analysis of slx-1;gen-1 double mutants revealed a 69% reduction in brood size (P<0.0001) and a 5.5-fold increase in larval arrest (P = 0.0079) compared with slx-1 single mutants. These results suggest that SLX-1 and GEN-1 share similar, albeit independent, mitotic roles at least during larval development.To investigate whether ffspring . A decreC. elegans germline facilitates the identification and analysis of specific stages of both mitotic and meiotic nuclei. Specifically, nuclei at the distal tip end are undergoing mitotic proliferation (zones 1 and 2), nuclei at the transition zone are in the leptotene/zygotene stages of meiosis (zone 3), followed by nuclei in early pachytene (zone 4), mid pachytene (zone 5) and late pachytene (zones 6 and 7) . Quantification of RAD-51 foci revealed that these were elevated in both mitotic (zones 1 and 2) and meiotic  nuclei in slx-1 mutants compared to wild type  . To inveild type . Moreovetic DSBs . Howeverild type . Increasin vivo, we performed a series of DNA damage sensitivity assays by exposing slx-1 mutants to γ-irradiation, which produces DSBs, nitrogen mustard (HN2), which produces DNA inter-strand crosslinks, camptothecin (CPT), which results in single-strand nicks, and UVC, which causes cyclobutane pyrimidine dimers  (him-18 (P<0.0001 and P<0.0001) and slx-1;him-18 (P = 0.0004 and P = 0.0002) double mutants after doses of either 50 or 100 Gy of irradiation  . In contadiation . These rslx-1 and slx-1;him-18 mutants share similar hypersensitivity to HN2 compared to wild type at both 100 µM and 200 µM, while him-18 mutants showed more severe hypersensitivity compared to slx-1 and slx-1;him-18 mutants , and meiotic RAD-51 foci peaked at 75 foci/nucleus at diplotene (−7 oocyte), only being completely absent by late-diakinesis (−1 oocyte)  by RAD-51 at the resected DSB ends is inhibited in the germline brc-2 mutants to that of RAD-51 foci (62.9) in rad-54 mutants in late pachytene nuclei (zone 7). Importantly, we confirmed that the elevated levels of RAD-51 foci we observe in rad-54 mutants at mid and late pachytene, where the events of repair we are focused on take place, are not already at a possible maximum thus obscuring our ability to utilize this mutant background to identify further increases in DSB levels. Specifically, following the formation of additional DSBs by γ-irradiation in rad-54 mutants, we observed 45 foci at 50 Gy compared to 29 foci at 0 Gy in zone 5 (P<0.0001)  . These t<0.0001) . slx-1rahenotype . Taken tC. elegans, as in many other species, crossover formation is biased towards the terminal thirds of autosomes slx-1 mutants, we performed three-dimensional traces of chromosomes in late pachytene nuclei by visualizing chromosome axes with an antibody to the meiotic cohesin REC-8 and quantified the levels and distribution of recombination intermediates along these chromosome axes with a RAD-51 antibody, comparing rad-54 and slx-1 rad-54 double mutants  of the autosomes  is higher  than in wild type, whereas it is reduced in the arm regions  , the frequency of crossovers in slx-1 is higher  than in wild type. These results suggest that SLX-1 is required for proper crossover distribution along both autosomes and the X chromosome. To further examine the role of SLX-1 in crossover distribution regulation, we measured crossover distribution on chromosomes IV and X in him-18 and slx-1;him-18 double mutants. Crossover levels are increased at the center of chromosome IV in both him-18 and slx-1;him-18 double mutants compared with wild type . Moreover, while the observed increases are similar between slx-1 and slx-1;him-18, only a moderate increase is detected in him-18 mutants on chromosome IV  . Using aild type . Crossovosome IV . Given tin vitro , the nucS. cerevisiae Zip3 protein, has been proposed to mark crossover precursor sites during late pachytene stage in C. elegansslx-1 mutants, we compared the numbers of ZHP-3-GFP foci present in pachytene nuclei in wild type and slx-1 mutants. The average number of ZHP-3-GFP foci observed in slx-1 mutants is 80% of those in wild type  (Flott and Brill 2003). In fission yeast, the Slx1 immunoprecipitation product cuts stem loops and HJs (symmetric cut) C. elegans, the preference of SLX-1-HIM-18 is for RFs>5′-flaps>HJs>3′-flaps   . Future n HIM-18 . This enn HIM-18 . Potentislx-1 mutants were hypersensitive to several kinds of DNA damaging agents  during the NER process C. elegans SLX-1 is longer than that of its homologs in other organisms, so it is possible that the N-terminal region confers the NER function of SLX-1. XPF is largely known as a repair factor for the NER pathway, including in C. elegansWe showed that g agents . Notablyslx-1 mutants  intermediates, and therefore earlier than dHJ formation bination .C. elegansC. elegans. One possibility is that the PHD finger of SLX-1 is involved in epigenetic change/read and somehow divides the arms and central regions along chromosomes  during early embryogenesis and the L3 larval stage in omosomes . It willTaken together, our analysis indicates that SLX-1 is required for several kinds of mitotic DNA repair pathways and reveals a role for this protein in the regulation of meiotic crossover distribution thereby promoting the maintenance of genomic integrity. Importantly, our study leads us to propose a model in which SLX-1 functions as a noncrossover promoting factor at the crossover cold regions during meiotic recombination.C. elegans strains were cultured at 20°C under standard conditions slx-1(tm2644), rad-54(ok615), hT2[bli-4(e937) let-?(q782) qIs48]; LGII: xpf-1(e1487), dpy-10(e128), unc-4(e120), mIn1[dpy-10(e128) mIs14](II); LGIII: him-18(tm2181), brc-2(tm1086), xnd-1(ok709), qC1[dpy-19(e1259) glp-1(q339) qIs26] (III); LGIV: pie-1::zhp-3::gfp, spo-11(ok79)P, him-6(ok412), rec-8(ok978), nT1[unc-?(n754) let-? qIs50] , nT1[qIs51] ; LGV: him-17(ok424), syp-2(ok307)opIs263 [Prpa-1::RPA-1-YFP::3′-UTR]slx-1 allele, tm2644, is predicted to encode for a catalytically inactive (nuclease-negative) protein. We also tried to knockdown slx-1 both by RNAi and by generating slx-1(tm2644)/Df trans-heterozygotes. RNAi utilizing feeding clones generated from either SLX-1 cDNA  mutant backgrounds. Attempts to generate a slx-1/Df trans-heterozygote for classical genetic characterization of the slx-1(tm2644) allele were hindered because the only available deficiency encompassing that gene, sDf4, involves a free chromosome duplication that interferes with this analysis.The HIM-18 and SLX-1 open reading frames in either pENTR or pDONR derivatives were transferred to the indicated expression vectors (pDEST-myc or pDEST-HA) using the Gateway cloning system (Invitrogen) and sequence validated. 293T cells were grown in Dulbecco Modified Eagle medium (DMEM) supplemented with 10% (v/v) FBS (Invitrogen), 100 units of penicillin per ml, and 0.1 mg streptomycin per ml. Antibodies against HA  and Myc  epitopes were utilized.32P-labeled DNA substrates  were prepared as previously described For protein interaction studies, the indicated proteins were expressed in HEK293T cells using Lipofectamine (Invitrogen) and after 24–48 h, cells were lysed in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 10 mM NaF, 1 mM EDTA+protease inhibitors (ROCHE), and cleared lysates used for immunoprecipitation with the indicated antibodies. Immune complexes were washed 4–5X with lysis buffer, re-suspended in SDS laemli buffer and were subjected to polyacrylamide gel separation and immunoblotting with the indicated antibodies. Recombinant GST-h.SLX1/His-h.SLX4(SBD) was purified as previously described Quantitative analysis of RAD-51 foci was performed as in C. elegans gonads were fixed and stained with rabbit α-RAD-51 (SDIX) , mouse α-REC-8 (Abcam) (1∶100) and guinea pig α-HIM-8 (1∶100). Chromosome axes were traced in 3D along the REC-8 signal and straightened by using either Priism 137 source at a dose rate of 1.86 Gy/min. For UVC sensitivity, animals were placed on the UV stratalinker 2400 (Stratagene) and exposed to 0, 300 or 600 J/m2. For nitrogen mustard (HN2) sensitivity, young adult animals were treated with 0, 100 or 200 µM of HN2  in M9 buffer containing E. coli OP50 with slow shaking in the dark for 19 hours. Treatment with camptothecin  was similar, but with doses of 0, 500 or 1000 nM. Following treatment with IR, UVC, HN2 or CPT, animals were plated to allow recovery for 3 hours. For all damage sensitivity experiments, 21 animals were plated 7 per plate and hatching was assessed for 4 hours after the recovery. After 1.5 days, hatched worms and dead eggs were counted. Each damage condition was replicated at least three times in independent experiments.To assess ionizing radiation (IR) sensitivity, animals (∼19 hours post L4 stage) were treated with 0, 50 or 100 Gy of IR from a Cs22–24 hour post-L4 hermaphrodites were stained with acridine orange (AO) for 2 hours and mounted under coverslips in 5 µl of a 15 mM sodium azide solution on 1.5% agarose pads. Apoptotic nuclei stained with AO were observed in the late pachytene region of the germline with a Leica DM5000 B fluorescence microscope. Between 33 and 47 gonads were scored for each genotype. Statistical comparisons between genotypes were performed using the two-tailed Mann-Whitney test, 95% C.I.DraI restriction digests of single worm lysates were performed as described in DraI SNP primers were utilized: A (uCE3-637), B (CE3-127), C (snp_Y39A1), D (uCE3-1426) for chromosome III, A (uCE4-515), B (pkP4055), C (snp_F49E11), D (pkP4099) for chromosome IV, A (pkP5076), B (snp_Y61A9L), C (pkP5129), D (snp_Y17D7B) for chromosome V, and A (pkP6143), B (pkP6105), B' (snp_F11A1), C (pkP6132), D (uCE6-1554) for the X chromosome. Statistical analysis was performed using the two-tailed Fisher's Exact test, 95% C.I., as in Meiotic crossover frequencies were assayed utilizing single-nucleotide polymorphisms (SNP) markers as in Recombination analysis using visible markers was performed as in 1–272 and SLX-1C273–443 were cloned into the Gateway donor vector (pDONR223). Each construct was then subcloned into 2 µ Gateway destination vectors pVV213 (activation domain (AD), LEU2+) and pVV212 , TRP1+). AD-Y and DB-X fusions were transformed into MATa Y8800 and MATa Y8930 yeast strains, respectively. These yeast strains have three reporter genes: GAL2-ADE2, met2::GAL7-lacZ and LYS2::GAL1-HIS3. MATa Y8800 and MATα Y8930 were mated on YPD plates and diploids carrying both plasmids were selected on SC-Leu-Trp plates. The interactions were assessed by growth on -His+1 mM 3-AT plates at 30°C.The yeast two-hybrid assay was performed according to Figure S132P-end-labeled 5′-flap, 3′-flap, HJ, or RF substrates, and the products were separated by native gel electrophoresis and visualized by autoradiography. The labeled substrates are indicated below the gel and the * indicates the labeled strand.Cleavage activity of HIM-18/SLX-1. The indicated proteins immuno-precipitated from 293T cells were incubated with (TIF)Click here for additional data file.Figure S2slx-1, him-18 and slx-1;him-18 mutants. Histograms depict the quantitation of RAD-51foci in germlines of the indicated genotypes. The number of RAD-51 foci per nucleus is categorized by the color code shown on the top. The percent of nuclei observed for each category (y-axis) are depicted for each zone along the germline axis (x-axis). 2–3 gonads were scored in each genotype. The number of RAD-51 foci per nucleus is categorized by the color code shown on the top. The percent of nuclei observed for each category (y-axis) are depicted for each zone along the germline axis (x-axis). 2–3 gonads were scored in each genotype.Mitotic and meiotic RAD-51 foci accumulate in (TIF)Click here for additional data file.Figure S3rad-54 mutants. Mean numbers of RAD-51 foci/nucleus are shown for rad-54 mutants following the indicated doses of exposure. Nuclei (N) from three gonads each were scored. Error bars indicate standard error of the mean.The levels of RAD-51 foci are not saturated in (TIF)Click here for additional data file.Figure S4brc-2 (tm1086) mutants. (A) Representative images of RPA-1-YFP localization in RPA-1-YFP and brc-2; RPA-1-YFP late pachytene nuclei (zone 7). Bars, 5 µm. (B) Mean numbers of RPA-1-YFP foci/nucleus are shown. Error bars indicate standard error of the mean.RPA-1-YFP foci accumulate in (TIF)Click here for additional data file.Figure S5slx-1 mutation in syp-2 and rec-8 mutants. (A) High magnification images of DAPI-stained bodies in the late diakinesis oocyte just before the spermatheca (−1 oocyte). The average number of DAPI-stained bodies is shown at the bottom right of each panel. Arrowheads indicate chromosome fragments. Bar, 1 µm. (B) Average number of DAPI-stained bodies including fragments. N = number of diakinesis nuclei scored for each genotype. Bars indicate standard error. NS indicates no statistical significance. (C) Quantitation of nuclei that contain at least one chromosome fragment. (D) Quantitation of DAPI-stained bodies.Chromosomal aberrations are not enhanced by (TIF)Click here for additional data file.Table S1DNA substrate oligonucleotides.(XLSX)Click here for additional data file.Table S2Number of RAD-51 foci/nucleus.(XLSX)Click here for additional data file.Table S3Summary of crossover distribution.(XLSX)Click here for additional data file.Table S4Statistical analysis of crossover distribution.(XLSX)Click here for additional data file.


Although most HTLV-1-infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% will develop either Adult T-cell Leukemia (ATL) or the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). To better understand differential gene expression in HTLV-1-associated disease, we examined the mRNA profiles of CD4+ T-cells isolated from ATL, HAM/TSP, AC and non-infected controls. Using genomic approaches, we identified gene signatures that discriminate between HTLV-1 clinical outcomes. Major activated cellular pathways included antimicrobial defense (KLRB1/SPN/SELPLG), innate immune sensing (TRAF3/AIM2/TLR2/TLR4/IKBKG/STAT3), cell adhesion and chemotaxis (CXCR4/CD2/CD63/CD48/CCL14/SPN/CCL13), thus supporting the idea of global immune disruption during HTLV-1 infection. Among the many genes identified, the suppressor of cytokine signaling 1, SOCS1, was upregulated in HAM/TSP and AC. SOCS1 expression positively correlated with high HTLV-1 mRNA load that is characteristic of HAM/TSP patients. SOCS1 inhibited cellular antiviral signaling during HTLV-1 infection by degrading IRF3, an essential transcription factor in the interferon pathway. This novel mechanism of viral evasion of the innate immune response can be directly related to the efficiency of HTLV-1 replication in patients with HAM/TSP. Despite SOCS1 ability to limit antiviral responses, CD4+ T-cells from HAM/TSP patients revealed a striking upregulation of IFN stimulated gene expression, viral host restriction factors and pro-inflammatory genes. We hypothesize that the magnitude of innate immune responses against HTLV-1 during de novo infection may predict clinical outcome; patients mounting a sustained/low antiviral response to HTLV-1 may remain AC with a low probability to progress to ATL, while those displaying an aberrant/high IFN response may progress to HAM/TSP.


Melanoma vaccines have been designed to expand specific CD8+ T-cells, but melanoma-reactive helper T-cells also can have antitumor activity. We previously observed clinical activity of a vaccine incorporating 6 melanoma helper peptides (6MHP), and found associations between CD4+ T cell response and survival. With the present study, in the spirit of personalized cancer immunotherapy, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides, and identify helper peptide-mediated augmentation of melanoma-specific CD8+ T-cell response.Thirty-seven patients with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund’s adjuvant. The vaccines contained 6 peptides: gp10044-59 (first 3 amino acids: WNR), Tyrosinase56-70 (AQN), Tyrosinase386-406 (FLL), Melan-A/MART-151-73 (RNG), MAGE-A3281-295 (TSY), and MAGE-A1, 2,3,6121-134 (LLK). Peripheral blood mononuclear cells (PBMC) and sentinel immunized nodes (SIN) were collected. CD4+ T-cell proliferation was assessed by thymidine uptake after exposure to peptides. CD8+ T-cell response was assessed by direct IFN-γ ELIspot assay against 14 MHC class I-restricted peptides.Vaccines induced CD4+ T cell responses to the 6MHP pool in 78% (29/37) of patients in SIN and 57% 21/37) in PBMC for an overall response rate of 81% (30/37), with responses to an average of 2 peptides per patient. The two most frequently immunogenic peptides were TSY at 49% (18/37) and FLL at 32% (12/37). HLA restriction was not limited to alleles originally described; for each peptide, the proportion of immune responsive patients whose HLA-DR expression matched those originally described was similar to the proportion of patients with different HLA-DR expression (unmatched, Figure /37 in PBThe 6MHP vaccine has CD4+ T-cell immunogenicity beyond known HLA-DR restrictions. Patients whose tumors express tyrosinase, MAGE-A3, and several other MAGE proteins may be ideal for vaccination with 6MHP. The 4 most immunogenic peptides warrant further study, perhaps in combination immune therapies.


Garrity1Oranmiyan W. NelsonThe purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.EuryarchaeotaPhylum Halobiforma lacisalsi AJ5, sequence accession AGFZ01000000 [Thermococcus sp. strain AM4, sequence accession CP002952 [ProteobacteriaPhylum Acidithiobacillus thiooxidans ATCC 19377, sequence accession AFOH01000000 [Acinetobacter sp. strain D499, sequence accession AGFH01000000 [Azospirillum brasilense Sp245, sequence accession HE577327 (chromosome), HE577328 (p1), HE577329 (p2), HE577330 (p3), HE577331 (p4), HE577332 (p5), HE577333 (p6) [Azospirillum lipoferum strain 4B, sequence accession FQ311868 (chromosome), FQ311869 (p1), FQ311870 (p2), FQ311871 (p3), FQ311872 (p4), FQ311873 (p5), FQ311874 (p6) [Q311873 p, FQ31187Escherichia coli F18, sequence accession AGTD01000000 [01000000 Escherichia coli K88, sequence accession CP002729 (chromosome), CP002730 (pUMNK88_K88), CP002731 (pUMNK88_IncI1), CP002732 (pUMNK88_Ent), CP002733 (pUMNK88_Hly), and HQ023862 (pUMNK88) [Escherichia coli Strain CE10, sequence accession CP003034 to CP003038 [CP003038 Gluconacetobacter xylinus, NBRC 3288, sequence accession AP012159 through AP012166 [Halomonas sp. Strain HAL1, sequence accession AGIB00000000 [00000000 Methylomonas methanica MC09, sequence accession CP002738 [CP002738 Novosphingobium nitrogenifigens Y88, sequence accession [ccession Pelagibacterium halotolerans B2, sequence accession CP003075 (chromosome), CP003076 (plasmid) [plasmid) Pseudomonas aeruginosa PAO1, sequence accession GSE34141 [GSE34141 Pseudomonas putida Idaho, sequence accession AGFJ01000000 [01000000 Sphingomonas elodea ATCC 31461, sequence accession AGFU01000000 [01000000 FirmicutesPhylum Acidaminococcus intestini RYC-MR95, sequence accession CP003058 [CP003058 Lactobacillus rhamnosus strain CASL, sequence accession AFYD00000000 [00000000 Pseudomonas aeruginosa NCGM2.S1, sequence accession AP012280 [AP012280 ActinobacteriaPhylum Candidatus Frankia datiscae, sequence accession NC_015656 [C_015656 Corynebacterium pseudotuberculosis Strain CIP 52.97, sequence accession CP003061 [CP003061 Propionibacterium acnes Type II Strain ATCC 11828, sequence accession CP003084 [CP003084 Streptomyces chartreusis NRRL 12338, sequence accession AGDE00000000 [00000000 Streptomyces chartreusis NRRL 3882, sequence accession AGDD00000000 [00000000 Streptomyces lysosuperificus ATCC 31396, accession number AGDC00000000 [00000000 Streptomyces sp. Strain Wigar10, sequence accession AGDF01000000 [01000000 SpirochaetesPhylum Borrelia afzelii PKo, sequence accession CP002933 (Chromosome), CP002942 (Ip17), CP002943 (Ip28-2), CP002944 (Ip28-3), CP002945 (Ip28-4), CP002946 (Ip28-7), CP002947 (IP28-8), CP002949 (Ip38), CP002950 (Ip54), CP002934 (cp26), CP002937 (cp32-1), CP002938 (cp32-3), CP002939 (cp32-5), CP002940 (cp32-7), CP002940 (cp32-9), CP002948 (cp32-10), CP002935 (cp32-11), CP002936 (cp32-12) [cp32-12) Borrelia afzelii ACA-1, sequence accession ABCU02000001-2 (Chromosome), CP001239 (Ip17), CP001238 (Ip28-1), CP001244 (Ip28-2), CP001241 (Ip28-3), CP001249 (Ip28-4), CP001242 (Ip-7), CP001246 (Ip38), CP001247 (Ip54), CP001250 (cp26), CP001243 (cp32-1), CP001237 (cp32-3), CP001240 (cp32-4), CP001248 (cp32-5), CP001245 (cp32-10) [Borrelia garinii PBr, sequence accession ABJV02000001-5 (Chromosome), CP001309 (Ip17), CP001301 (Ip25), CP001310 (Ip28-1), CP001307 (Ip28-3), CP001304 (Ip28-4), CP001311 (Ip28-7), CP001302 (Ip36), CP001308 (Ip54), CP001305 (Ip26), CP001303 (Ip32-5), CP001306 (Ip32-10) [Ip32-10) Borrelia garinii Far 04, sequence accession ABPZ02000001-33 (Chromosome), CP001315 (Ip17), CP001317 (Ip25), CP001316 (Ip28-1), CP001314 (Ip36), CP001318 (Ip54), CP001319 (Ip26), CP001320 (Ip32-10) [Ip32-10) Non-Bacterial genomesB1 Human Adenovirus HAdV-16 strain E26, sequence accession JN860680 B1 Human Adenovirus HAdV-3/16, sequence accession JN860678 B1 Human Adenovirus HAdV-3+7, sequence accession JN860679 B1 Human Adenovirus HAdV-7d2, sequence accession JN860677 B1 Human Adenovirus HAdV-7h, sequence accession JN860676 Bacillus cereus bacteriophage BCP78, sequence accession JN797797 [JN797797 Circoviridae member , sequence accession JF803741 Coccolithovirus Emiliania huxleyi Virus 203, sequence accession JF974291 Cryptococcus gattii BC, sequence accession SRP006436 [RP006436 Erwinia amylovora plasmid pEI70, sequence accession CP002951 Mortierella alpina, sequence accession ADAG00000000 [00000000 Parvovirus Aj-BtPV-1, sequence accession JN860679 Parvovirus Eh-BtPV-1, sequence accession JN860679 Penicillium marneffei PM1, sequence accession AGCC00000000 Pseudomonas fluorescens phage OBP, sequence accession JN627160 Salmonella bacteriophage SPN3US, sequence accession JN641803 Tailam virus, sequence accession JN689227