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Epigenetic Regulation of Antibody Responses by the Histone H2A Deubiquitinase MYSM1.

Jiang XX, Chou Y, Jones L, Wang T, Sanchez S, Huang XF, Zhang L, Wang C, Chen SY - Sci Rep (2015)

Bottom Line: In this study, we found that mice deficient in the histone H2A deubiquitinase MYSM1, despite their severe defect in B cell development, exhibit an enhanced antibody response against both T cell-dependent and independent antigens.Mechanistic studies demonstrated that MYSM1 is a transcriptional activator of Pax5, the repressors of plasma cell differentiation, by facilitating key transcriptional factor recruitment and coordinating histone modifications at the Pax5 loci.Importantly, this study further provides a new target and strategy to modulate antibody production and responses with profound therapeutic implications.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Norris Comprehensive Cancer Center Keck School of Medicine, University of Southern California, Los Angeles, California, 90033, USA.

ABSTRACT
B cell-mediated antibody response plays critical roles in protective immunity, as well as in the pathogenesis of allergic and autoimmune diseases. Epigenetic histone and DNA modifications regulate gene transcription and immunity; however, so far, little is known about the role of epigenetic regulation in antibody responses. In this study, we found that mice deficient in the histone H2A deubiquitinase MYSM1, despite their severe defect in B cell development, exhibit an enhanced antibody response against both T cell-dependent and independent antigens. We revealed that MYSM1 intrinsically represses plasma cell differentiation and antibody production. Mechanistic studies demonstrated that MYSM1 is a transcriptional activator of Pax5, the repressors of plasma cell differentiation, by facilitating key transcriptional factor recruitment and coordinating histone modifications at the Pax5 loci. Hence, this study uncovers a critical role for MYSM1 in epigenetically repressing plasma cell differentiation and antibody production, in addition to its opposing, active role in B cell development. Importantly, this study further provides a new target and strategy to modulate antibody production and responses with profound therapeutic implications.

No MeSH data available.


Related in: MedlinePlus

MYSM1 interacts with the transcription factor PU.1 for recruitment to the Pax5 locus. (a,b) Sequential two-step ChIP assays of naïve splenic WT B220+ cells (a) and WT CD138+B220− plasma cells (b) were performed, showing the recruitment of the endogenous PU.1 and MYSM1 to the Pax5 promoter in naïve WT B cells, but not in WT plasma cells, from one of two independent experiments. The relative binding was defined by determining the immunoprecipitation level (ratio of the amount of immunoprecipitated DNA to that of the input sample) and then comparing to corresponding first ChIP or second ChIP control IgG immunoprecipitation level, which was set as 1.0. **P < 0.01, IgG vs. MYSM1, *P < 0.05, IgG vs. PU.1. (c) Co-immunoprecipitation of PU.1 and MYSM1. Cell lysates from splenic WT B cells transduced with lentivirus containing Mysm1 vector (LV-MYSM1) or control flag vector (LV-Flag) were immunoprecipitated with anti-Flag antibody, then probed with a PU.1 antibody. Five percent of the cell lysate input was loaded. (d) Drastic reduction of PU.1 occupancy at the Pax5 promotion region of naïve Mysm1−/− B cells, compared to that of naïve WT B cells. ChIP data are presented from one of two independent experiments. **P < 0.01, WT vs. Mysm1−/−. (e) Altered histone modifications at the Pax5 locus in splenic Mysm1−/− B cells. ChIP analysis of naïve splenic WT or Mysm1−/− B cells. The DNA precipitated with the indicated antibodies was analyzed by quantitative PCR with primers amplifying the Pax5 promoter region and normalized with input DNA before being compared to WT (set as 1). Data are presented from one of two independent experiments. **P < 0.01, *P < 0.05, WT vs. Mysm1−/−.
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f7: MYSM1 interacts with the transcription factor PU.1 for recruitment to the Pax5 locus. (a,b) Sequential two-step ChIP assays of naïve splenic WT B220+ cells (a) and WT CD138+B220− plasma cells (b) were performed, showing the recruitment of the endogenous PU.1 and MYSM1 to the Pax5 promoter in naïve WT B cells, but not in WT plasma cells, from one of two independent experiments. The relative binding was defined by determining the immunoprecipitation level (ratio of the amount of immunoprecipitated DNA to that of the input sample) and then comparing to corresponding first ChIP or second ChIP control IgG immunoprecipitation level, which was set as 1.0. **P < 0.01, IgG vs. MYSM1, *P < 0.05, IgG vs. PU.1. (c) Co-immunoprecipitation of PU.1 and MYSM1. Cell lysates from splenic WT B cells transduced with lentivirus containing Mysm1 vector (LV-MYSM1) or control flag vector (LV-Flag) were immunoprecipitated with anti-Flag antibody, then probed with a PU.1 antibody. Five percent of the cell lysate input was loaded. (d) Drastic reduction of PU.1 occupancy at the Pax5 promotion region of naïve Mysm1−/− B cells, compared to that of naïve WT B cells. ChIP data are presented from one of two independent experiments. **P < 0.01, WT vs. Mysm1−/−. (e) Altered histone modifications at the Pax5 locus in splenic Mysm1−/− B cells. ChIP analysis of naïve splenic WT or Mysm1−/− B cells. The DNA precipitated with the indicated antibodies was analyzed by quantitative PCR with primers amplifying the Pax5 promoter region and normalized with input DNA before being compared to WT (set as 1). Data are presented from one of two independent experiments. **P < 0.01, *P < 0.05, WT vs. Mysm1−/−.

Mentions: MYSM1 was found to regulate target gene transcription by directing histone H2A deubiquitination and additional histone modifications, and transcription factor recruitment to target loci21. Several transcription factors, such as PU.1, EBF1, IRF4, and NF-kB, were found to activate Pax5 transcription in B cells30. To investigate whether MYSM1 interacts with the transcriptional factors of Pax5 at the Pax5 locus, we performed a sequential two-step ChIP assay with the first anti-MYSM1 ChIP, followed by the second ChIP with one of the antibodies against these known transcription activators for Pax5 transcription21. Through the sequential two-step ChIP assays, we found that MYSM1 was associated with PU.1 at the Pax5 locus in naïve WT B cells (Fig. 7a). However, the association of MYSM1 with IRF4 (Fig. 6b), Stat3/5, NF-kB, and AP-1 at the Pax5 locus in naïve WT B cells was not positively identified in our assays. Consistent with earlier observations (Fig. 6b), the association of MYSM1 and PU.1 with the Pax5 locus was not detected in WT plasma cells. We then used co-immunoprecipitation assays to confirm the association. MYSM1 protein was found to co-precipitate with endogenous PU.1 proteins in naïve WT B cells (Fig. 7c). Furthermore, we tested whether MYSM1 is required for the recruitment of PU.1 to the Pax5 locus in naïve B cells by ChIP assays of naïve WT and Mysm1−/− B cells. Figure 7d shows that the transcription factor PU.1 was associated with the Pax5 locus in WT B cells, but this association was not detected in Mysm1−/− B cells, indicating a critical role of MYSM1 in the recruitment of PU.1 to the Pax5 locus in naïve B cells. Moreover, we used ChIP assays with antibodies against various histone markers to investigate whether histone modifications at the target Pax5 locus were altered in Mysm1−/− B cells. We observed an increase in ubH2A levels at the Pax5 promoter region of Mysm1−/− B cells (Fig. 7e). We also examined the levels of representative histone modifications that are known to be associated with transcriptional activation (H3K4me3 and H3K9ac) or repression (H3K27me3 and H3K9me3), and saw an increase in the levels of repressive marks (H3K27me3) and a decrease in the levels of active marks (H3K4me3 and H3K9ac) at the Pax5 locus of the Mysm1−/− B cells as compared to WT B cells (Fig. 7e). Together, these data indicate that MYSM1 likely plays a critical role in regulating histone modifications at the target Pax5 locus and that MYSM1 is required for the recruitment of the transcription factor PU.1 to the Pax5 locus in naïve B cells.


Epigenetic Regulation of Antibody Responses by the Histone H2A Deubiquitinase MYSM1.

Jiang XX, Chou Y, Jones L, Wang T, Sanchez S, Huang XF, Zhang L, Wang C, Chen SY - Sci Rep (2015)

MYSM1 interacts with the transcription factor PU.1 for recruitment to the Pax5 locus. (a,b) Sequential two-step ChIP assays of naïve splenic WT B220+ cells (a) and WT CD138+B220− plasma cells (b) were performed, showing the recruitment of the endogenous PU.1 and MYSM1 to the Pax5 promoter in naïve WT B cells, but not in WT plasma cells, from one of two independent experiments. The relative binding was defined by determining the immunoprecipitation level (ratio of the amount of immunoprecipitated DNA to that of the input sample) and then comparing to corresponding first ChIP or second ChIP control IgG immunoprecipitation level, which was set as 1.0. **P < 0.01, IgG vs. MYSM1, *P < 0.05, IgG vs. PU.1. (c) Co-immunoprecipitation of PU.1 and MYSM1. Cell lysates from splenic WT B cells transduced with lentivirus containing Mysm1 vector (LV-MYSM1) or control flag vector (LV-Flag) were immunoprecipitated with anti-Flag antibody, then probed with a PU.1 antibody. Five percent of the cell lysate input was loaded. (d) Drastic reduction of PU.1 occupancy at the Pax5 promotion region of naïve Mysm1−/− B cells, compared to that of naïve WT B cells. ChIP data are presented from one of two independent experiments. **P < 0.01, WT vs. Mysm1−/−. (e) Altered histone modifications at the Pax5 locus in splenic Mysm1−/− B cells. ChIP analysis of naïve splenic WT or Mysm1−/− B cells. The DNA precipitated with the indicated antibodies was analyzed by quantitative PCR with primers amplifying the Pax5 promoter region and normalized with input DNA before being compared to WT (set as 1). Data are presented from one of two independent experiments. **P < 0.01, *P < 0.05, WT vs. Mysm1−/−.
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f7: MYSM1 interacts with the transcription factor PU.1 for recruitment to the Pax5 locus. (a,b) Sequential two-step ChIP assays of naïve splenic WT B220+ cells (a) and WT CD138+B220− plasma cells (b) were performed, showing the recruitment of the endogenous PU.1 and MYSM1 to the Pax5 promoter in naïve WT B cells, but not in WT plasma cells, from one of two independent experiments. The relative binding was defined by determining the immunoprecipitation level (ratio of the amount of immunoprecipitated DNA to that of the input sample) and then comparing to corresponding first ChIP or second ChIP control IgG immunoprecipitation level, which was set as 1.0. **P < 0.01, IgG vs. MYSM1, *P < 0.05, IgG vs. PU.1. (c) Co-immunoprecipitation of PU.1 and MYSM1. Cell lysates from splenic WT B cells transduced with lentivirus containing Mysm1 vector (LV-MYSM1) or control flag vector (LV-Flag) were immunoprecipitated with anti-Flag antibody, then probed with a PU.1 antibody. Five percent of the cell lysate input was loaded. (d) Drastic reduction of PU.1 occupancy at the Pax5 promotion region of naïve Mysm1−/− B cells, compared to that of naïve WT B cells. ChIP data are presented from one of two independent experiments. **P < 0.01, WT vs. Mysm1−/−. (e) Altered histone modifications at the Pax5 locus in splenic Mysm1−/− B cells. ChIP analysis of naïve splenic WT or Mysm1−/− B cells. The DNA precipitated with the indicated antibodies was analyzed by quantitative PCR with primers amplifying the Pax5 promoter region and normalized with input DNA before being compared to WT (set as 1). Data are presented from one of two independent experiments. **P < 0.01, *P < 0.05, WT vs. Mysm1−/−.
Mentions: MYSM1 was found to regulate target gene transcription by directing histone H2A deubiquitination and additional histone modifications, and transcription factor recruitment to target loci21. Several transcription factors, such as PU.1, EBF1, IRF4, and NF-kB, were found to activate Pax5 transcription in B cells30. To investigate whether MYSM1 interacts with the transcriptional factors of Pax5 at the Pax5 locus, we performed a sequential two-step ChIP assay with the first anti-MYSM1 ChIP, followed by the second ChIP with one of the antibodies against these known transcription activators for Pax5 transcription21. Through the sequential two-step ChIP assays, we found that MYSM1 was associated with PU.1 at the Pax5 locus in naïve WT B cells (Fig. 7a). However, the association of MYSM1 with IRF4 (Fig. 6b), Stat3/5, NF-kB, and AP-1 at the Pax5 locus in naïve WT B cells was not positively identified in our assays. Consistent with earlier observations (Fig. 6b), the association of MYSM1 and PU.1 with the Pax5 locus was not detected in WT plasma cells. We then used co-immunoprecipitation assays to confirm the association. MYSM1 protein was found to co-precipitate with endogenous PU.1 proteins in naïve WT B cells (Fig. 7c). Furthermore, we tested whether MYSM1 is required for the recruitment of PU.1 to the Pax5 locus in naïve B cells by ChIP assays of naïve WT and Mysm1−/− B cells. Figure 7d shows that the transcription factor PU.1 was associated with the Pax5 locus in WT B cells, but this association was not detected in Mysm1−/− B cells, indicating a critical role of MYSM1 in the recruitment of PU.1 to the Pax5 locus in naïve B cells. Moreover, we used ChIP assays with antibodies against various histone markers to investigate whether histone modifications at the target Pax5 locus were altered in Mysm1−/− B cells. We observed an increase in ubH2A levels at the Pax5 promoter region of Mysm1−/− B cells (Fig. 7e). We also examined the levels of representative histone modifications that are known to be associated with transcriptional activation (H3K4me3 and H3K9ac) or repression (H3K27me3 and H3K9me3), and saw an increase in the levels of repressive marks (H3K27me3) and a decrease in the levels of active marks (H3K4me3 and H3K9ac) at the Pax5 locus of the Mysm1−/− B cells as compared to WT B cells (Fig. 7e). Together, these data indicate that MYSM1 likely plays a critical role in regulating histone modifications at the target Pax5 locus and that MYSM1 is required for the recruitment of the transcription factor PU.1 to the Pax5 locus in naïve B cells.

Bottom Line: In this study, we found that mice deficient in the histone H2A deubiquitinase MYSM1, despite their severe defect in B cell development, exhibit an enhanced antibody response against both T cell-dependent and independent antigens.Mechanistic studies demonstrated that MYSM1 is a transcriptional activator of Pax5, the repressors of plasma cell differentiation, by facilitating key transcriptional factor recruitment and coordinating histone modifications at the Pax5 loci.Importantly, this study further provides a new target and strategy to modulate antibody production and responses with profound therapeutic implications.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Norris Comprehensive Cancer Center Keck School of Medicine, University of Southern California, Los Angeles, California, 90033, USA.

ABSTRACT
B cell-mediated antibody response plays critical roles in protective immunity, as well as in the pathogenesis of allergic and autoimmune diseases. Epigenetic histone and DNA modifications regulate gene transcription and immunity; however, so far, little is known about the role of epigenetic regulation in antibody responses. In this study, we found that mice deficient in the histone H2A deubiquitinase MYSM1, despite their severe defect in B cell development, exhibit an enhanced antibody response against both T cell-dependent and independent antigens. We revealed that MYSM1 intrinsically represses plasma cell differentiation and antibody production. Mechanistic studies demonstrated that MYSM1 is a transcriptional activator of Pax5, the repressors of plasma cell differentiation, by facilitating key transcriptional factor recruitment and coordinating histone modifications at the Pax5 loci. Hence, this study uncovers a critical role for MYSM1 in epigenetically repressing plasma cell differentiation and antibody production, in addition to its opposing, active role in B cell development. Importantly, this study further provides a new target and strategy to modulate antibody production and responses with profound therapeutic implications.

No MeSH data available.


Related in: MedlinePlus