RESUMO
Deregulated overexpression of MYC is implicated in the development and malignant progression of most (â¼70%) human tumors. MYC drives cell growth and proliferation, but also, at high levels, promotes apoptosis. Here, we report that the proliferative capacity of MYC-driven normal and neoplastic B lymphoid cells depends on MNT, a MYC-related transcriptional repressor. Our genetic data establish that MNT synergizes with MYC by suppressing MYC-driven apoptosis, and that it does so primarily by reducing the level of pro-apoptotic BIM. In Eµ-Myc mice, which model the MYC/IGH chromosome translocation in Burkitt's lymphoma, homozygous Mnt deletion greatly reduced lymphoma incidence by enhancing apoptosis and markedly decreasing premalignant B lymphoid cell populations. Strikingly, by inducing Mnt deletion within transplanted fully malignant Eµ-Myc lymphoma cells, we significantly extended transplant recipient survival. The dependency of lymphomas on MNT for survival suggests that drugs inhibiting MNT could significantly boost therapy of MYC-driven tumors by enhancing intrinsic MYC-driven apoptosis.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Transformação Celular Neoplásica/genética , Linfoma/genética , Linfoma/mortalidade , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Antineoplásicos/uso terapêutico , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Medula Óssea/metabolismo , Medula Óssea/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Linfoma/tratamento farmacológico , Linfoma/patologia , Linfoma de Células B/genética , Camundongos , Camundongos Transgênicos , Proteínas Repressoras/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In B cells, Ig class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), the activity of which leads to DNA double-strand breaks (DSBs) within IgH switch (S) regions. Preferential targeting of AID-mediated DSBs to S sequences is critical for allowing diversification of antibody functions, while minimizing potential off-target oncogenic events. Here, we used gene targeted inactivation of histone methyltransferase (HMT) multiple myeloma SET domain (MMSET) in mouse B cells and the CH12F3 cell line to explore its role in CSR. We find that deletion of MMSET-II, the isoform containing the catalytic SET domain, inhibits CSR without affecting either IgH germline transcription or joining of DSBs within S regions by classical nonhomologous end joining (C-NHEJ). Instead, we find that MMSET-II inactivation leads to decreased AID recruitment and DSBs at the upstream donor Sµ region. Our findings suggest a role for the HMT MMSET in promoting AID-mediated DNA breaks during CSR.
Assuntos
Citidina Desaminase/genética , DNA/genética , Histona-Lisina N-Metiltransferase/genética , Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Imunoglobulinas/genética , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Domínio Catalítico , Citidina Desaminase/imunologia , DNA/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Regulação da Expressão Gênica , Inativação Gênica , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/imunologia , Imunoglobulinas/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/imunologia , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Recombinação Genética , Transdução de SinaisRESUMO
In response to antigens and cytokines, mouse B cells undergo class-switch recombination (CSR) and differentiate into Ig-secreting cells. T-bet, a T-box transcription factor that is up-regulated in lymphocytes by IFN-γ or IL-27, was shown to regulate CSR to IgG2a after T cell-independent B-cell stimulations. However, the molecular mechanisms controlling this process remain unclear. In the present study, we show that inactivation of the Ets-1 transcription factor results in a severe decrease in IgG2a secretion in vivo and in vitro. No T-bet expression was observed in Ets-1-deficient (Ets-1(-/-)) B cells stimulated with IFN-γ and lipopolysaccharide, and forced expression of T-bet in these cells rescued IgG2a secretion. Furthermore, we identified a transcriptional enhancer in the T-bet locus with an activity in B cells that relies on ETS-binding sites. After IFN-γ stimulation of Ets-1(-/-) B cells, activated Stat1, which forms a complex with Ets-1 in wild-type cells, no longer binds to the T-bet enhancer or promotes histone modifications at this site. These results demonstrate that Ets-1 is critical for IgG2a CSR and acts as an essential cofactor for Stat1 in the regulation of T-bet expression in B cells.
Assuntos
Linfócitos B/metabolismo , Switching de Imunoglobulina/fisiologia , Imunoglobulina G/biossíntese , Proteína Proto-Oncogênica c-ets-1/fisiologia , Fator de Transcrição STAT1/fisiologia , Proteínas com Domínio T/fisiologia , Acetilação , Animais , Linfócitos B/imunologia , Proteínas de Ligação a DNA/deficiência , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Histonas/metabolismo , Switching de Imunoglobulina/genética , Interferon gama/farmacologia , Subunidade gama Comum de Receptores de Interleucina/deficiência , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Processamento de Proteína Pós-Traducional , Proteína Proto-Oncogênica c-ets-1/deficiência , Proteína Proto-Oncogênica c-ets-1/genética , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Organismos Livres de Patógenos Específicos , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genéticaRESUMO
The importance of c-MYC in regulating lymphopoiesis and promoting lymphomagenesis is well-established. Far less appreciated is the vital supporting role of MYC's relative MNT. Using Rag1Cre-mediated Mnt deletion in lymphoid progenitor cells, we show here that, during normal T cell development, MNT loss enhances apoptosis, at least in part by elevating expression of the pro-apoptotic BH3-only protein BIM. Moreover, using T lymphoma-prone VavP-MYC transgenic mice, we show that Mnt deletion reduces the pool of pre-malignant MYC-driven T lymphoid cells and abrogates thymic T lymphomagenesis. In addition, we establish that Mnt deletion prevents T lymphoma development in γ-irradiated mice, most likely by enhancing apoptosis of T lymphoid cells repopulating the depleted thymus. Taken together with our recent demonstration that MNT is vital for the survival of MYC-driven pre-malignant and malignant B lymphoid cells, these results suggest that MNT represents an important new drug target for both T and B lymphoid malignancies.
Assuntos
Apoptose , Linfoma , Animais , Camundongos , Linfócitos/metabolismo , Linfoma/genética , Linfoma/patologia , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linfócitos T/metabolismoRESUMO
Regulatory T cells (T reg cells) constitute a population of CD4(+) T cells that limits immune responses. The transcription factor Foxp3 is important for determining the development and function of T reg cells; however, the molecular mechanisms that trigger and maintain its expression remain incompletely understood. In this study, we show that mice deficient for the Ets-1 transcription factor (Ets-1(-/-)) developed T cell-mediated splenomegaly and systemic autoimmunity that can be blocked by functional wild-type T reg cells. Spleens of Ets-1(-/-) mice contained mostly activated T cells, including Th2-polarized CD4(+) cells and had reduced percentages of T reg cells. Splenic and thymic Ets-1(-/-) T reg cells expressed low levels of Foxp3 and displayed the CD103 marker that characterizes antigen-experienced T reg cells. Thymic development of Ets-1(-/-) T reg cells appeared intrinsically altered as Foxp3-expressing cells differentiate poorly in mixed fetal liver reconstituted chimera and fetal thymic organ culture. Ets-1(-/-) T reg cells showed decreased in vitro suppression activity and did not protect Rag2(-/-) hosts from naive T cell-induced inflammatory bowel disease. Furthermore, in T reg cells, Ets-1 interacted with the Foxp3 intronic enhancer and was required for demethylation of this regulatory sequence. These data demonstrate that Ets-1 is required for the development of natural T reg cells and suggest a role for this transcription factor in the regulation of Foxp3 expression.