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is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}

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is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}

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is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}

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is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}

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is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}

{"project":"GO-BP","denotations":[{"id":"T5069","span":{"begin":88,"end":101},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T5070","span":{"begin":2355,"end":2368},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T5071","span":{"begin":171,"end":191},"obj":"http://purl.obolibrary.org/obo/GO_0033580"},{"id":"T5072","span":{"begin":171,"end":191},"obj":"http://purl.obolibrary.org/obo/GO_0034394"},{"id":"T5073","span":{"begin":171,"end":191},"obj":"http://purl.obolibrary.org/obo/GO_0033575"},{"id":"T5074","span":{"begin":328,"end":337},"obj":"http://purl.obolibrary.org/obo/GO_0023052"},{"id":"T5075","span":{"begin":2797,"end":2806},"obj":"http://purl.obolibrary.org/obo/GO_0023052"},{"id":"T5076","span":{"begin":1475,"end":1506},"obj":"http://purl.obolibrary.org/obo/GO_0045911"},{"id":"T5077","span":{"begin":1489,"end":1506},"obj":"http://purl.obolibrary.org/obo/GO_0006310"},{"id":"T5078","span":{"begin":1597,"end":1612},"obj":"http://purl.obolibrary.org/obo/GO_0045190"},{"id":"T5079","span":{"begin":1603,"end":1624},"obj":"http://purl.obolibrary.org/obo/GO_0045190"},{"id":"T5080","span":{"begin":1597,"end":1624},"obj":"http://purl.obolibrary.org/obo/GO_0048290"},{"id":"T5081","span":{"begin":1597,"end":1624},"obj":"http://purl.obolibrary.org/obo/GO_0048292"},{"id":"T5082","span":{"begin":1597,"end":1624},"obj":"http://purl.obolibrary.org/obo/GO_0048289"},{"id":"T5083","span":{"begin":1597,"end":1624},"obj":"http://purl.obolibrary.org/obo/GO_0048291"},{"id":"T5084","span":{"begin":1778,"end":1793},"obj":"http://purl.obolibrary.org/obo/GO_0006955"},{"id":"T5085","span":{"begin":2770,"end":2778},"obj":"http://purl.obolibrary.org/obo/GO_0007349"},{"id":"T15595","span":{"begin":1184,"end":1196},"obj":"http://purl.obolibrary.org/obo/GO_0009293"},{"id":"T15596","span":{"begin":1318,"end":1330},"obj":"http://purl.obolibrary.org/obo/GO_0009293"},{"id":"T16041","span":{"begin":2856,"end":2869},"obj":"http://purl.obolibrary.org/obo/GO_0006351"}],"text":"HOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}

{"project":"GO-MF","denotations":[{"id":"T5086","span":{"begin":1514,"end":1528},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T5087","span":{"begin":1582,"end":1596},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T5088","span":{"begin":1735,"end":1743},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T5089","span":{"begin":2294,"end":2302},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T5090","span":{"begin":2525,"end":2533},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T5091","span":{"begin":2662,"end":2670},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T5092","span":{"begin":1849,"end":1853},"obj":"http://purl.obolibrary.org/obo/GO_0005136"},{"id":"T5093","span":{"begin":2205,"end":2209},"obj":"http://purl.obolibrary.org/obo/GO_0005136"},{"id":"T5094","span":{"begin":2338,"end":2342},"obj":"http://purl.obolibrary.org/obo/GO_0005136"},{"id":"T5095","span":{"begin":2557,"end":2561},"obj":"http://purl.obolibrary.org/obo/GO_0005136"},{"id":"T5096","span":{"begin":2694,"end":2698},"obj":"http://purl.obolibrary.org/obo/GO_0005136"},{"id":"T15597","span":{"begin":976,"end":984},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T15598","span":{"begin":1005,"end":1013},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T15599","span":{"begin":1112,"end":1120},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T15600","span":{"begin":1173,"end":1181},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T16042","span":{"begin":3204,"end":3212},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T16043","span":{"begin":3236,"end":3240},"obj":"http://purl.obolibrary.org/obo/GO_0005136"}],"text":"HOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}

{"project":"GO-CC","denotations":[{"id":"T16044","span":{"begin":2917,"end":2922},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16045","span":{"begin":2950,"end":2955},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16046","span":{"begin":3067,"end":3072},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16047","span":{"begin":3268,"end":3273},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16048","span":{"begin":3204,"end":3212},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T16049","span":{"begin":3204,"end":3212},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T5097","span":{"begin":126,"end":131},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5098","span":{"begin":222,"end":227},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5099","span":{"begin":247,"end":252},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5100","span":{"begin":464,"end":469},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5101","span":{"begin":604,"end":609},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5102","span":{"begin":687,"end":692},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5103","span":{"begin":764,"end":769},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5104","span":{"begin":1442,"end":1447},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5105","span":{"begin":1646,"end":1651},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5106","span":{"begin":2388,"end":2393},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5107","span":{"begin":2486,"end":2491},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5108","span":{"begin":2630,"end":2635},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T5109","span":{"begin":171,"end":183},"obj":"http://purl.obolibrary.org/obo/GO_0009986"},{"id":"T5110","span":{"begin":263,"end":266},"obj":"http://purl.obolibrary.org/obo/GO_0005680"},{"id":"T5111","span":{"begin":1735,"end":1743},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T5112","span":{"begin":2294,"end":2302},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T5113","span":{"begin":2525,"end":2533},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T5114","span":{"begin":2662,"end":2670},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T5115","span":{"begin":1735,"end":1743},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T5116","span":{"begin":2294,"end":2302},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T5117","span":{"begin":2525,"end":2533},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T5118","span":{"begin":2662,"end":2670},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T15601","span":{"begin":913,"end":918},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T15602","span":{"begin":976,"end":984},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T15603","span":{"begin":1005,"end":1013},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T15604","span":{"begin":1112,"end":1120},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T15605","span":{"begin":1173,"end":1181},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T15606","span":{"begin":976,"end":984},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T15607","span":{"begin":1005,"end":1013},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T15608","span":{"begin":1112,"end":1120},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T15609","span":{"begin":1173,"end":1181},"obj":"http://purl.obolibrary.org/obo/GO_0042571"}],"text":"HOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}

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Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}

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is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nH"}