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1.
Mol Cell Biol ; 39(13)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30988160

RESUMO

Growth factor independence 1B (GFI1B) coordinates assembly of transcriptional repressor complexes comprised of corepressors and histone-modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B's obligate effector, lysine-specific demethylase 1 (LSD1). We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin-remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2, and ZNF217) in an LSD1-dependent manner to control acquisition of erythroid traits by K562 cells. Among these elements, depletion of both HMG20A and HMG20B or of GSE1 blocks GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B-LSD1 binding. These findings demonstrate the central role of the GFI1B-LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.


Assuntos
Células Eritroides/citologia , Histona Desmetilases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células COS , Diferenciação Celular , Chlorocebus aethiops , Regulação para Baixo , Células Eritroides/metabolismo , Histona Desacetilases/metabolismo , Humanos , Células K562 , Fenótipo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Genética
3.
Biochem J ; 473(19): 3355-69, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27480105

RESUMO

Proper hematopoietic cell fate decisions require co-ordinated functions of transcription factors, their associated co-regulators, and histone-modifying enzymes. Growth factor independence 1 (GFI1) is a zinc finger transcriptional repressor and master regulator of normal and malignant hematopoiesis. While several GFI1-interacting proteins have been described, how GFI1 leverages these relationships to carry out transcriptional repression remains unclear. Here, we describe a functional axis involving GFI1, SMYD2, and LSD1 that is a critical contributor to GFI1-mediated transcriptional repression. SMYD2 methylates lysine-8 (K8) within a -(8)KSKK(11)- motif embedded in the GFI1 SNAG domain. Methylation-defective GFI1 SNAG domain lacks repressor function due to failure of LSD1 recruitment and persistence of promoter H3K4 di-methyl marks. Methylation-defective GFI1 also fails to complement GFI1 depletion phenotypes in developing zebrafish and lacks pro-growth and survival functions in lymphoid leukemia cells. Our data show a discrete methylation event in the GFI1 SNAG domain that facilitates recruitment of LSD1 to enable transcriptional repression and co-ordinate control of hematopoietic cell fate in both normal and malignant settings.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Histona Desmetilases/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Genética/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem da Célula , Metilação de DNA , Proteínas de Ligação a DNA/química , Humanos , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Peixe-Zebra
4.
Mol Cell Biol ; 36(10): 1438-50, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26951200

RESUMO

Cell fate specification requires precise coordination of transcription factors and their regulators to achieve fidelity and flexibility in lineage allocation. The transcriptional repressor growth factor independence 1 (GFI1) is comprised of conserved Snail/Slug/Gfi1 (SNAG) and zinc finger motifs separated by a linker region poorly conserved with GFI1B, its closest homolog. Moreover, GFI1 and GFI1B coordinate distinct developmental fates in hematopoiesis, suggesting that their functional differences may derive from structures within their linkers. We show a binding interface between the GFI1 linker and the SP-RING domain of PIAS3, an E3-SUMO (small ubiquitin-related modifier) ligase. The PIAS3 binding region in GFI1 contains a conserved type I SUMOylation consensus element, centered on lysine-239 (K239). In silico prediction algorithms identify K239 as the only high-probability site for SUMO modification. We show that GFI1 is modified by SUMO at K239. SUMOylation-resistant derivatives of GFI1 fail to complement Gfi1 depletion phenotypes in zebrafish primitive erythropoiesis and granulocytic differentiation in cultured human cells. LSD1/CoREST recruitment and MYC repression by GFI1 are profoundly impaired for SUMOylation-resistant GFI1 derivatives, while enforced expression of MYC blocks granulocytic differentiation. These findings suggest that SUMOylation within the GFI1 linker favors LSD1/CoREST recruitment and MYC repression to govern hematopoietic differentiation.


Assuntos
Hematopoese , Histona Desmetilases/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Células COS , Diferenciação Celular , Chlorocebus aethiops , Regulação da Expressão Gênica , Células HEK293 , Células HL-60 , Humanos , Lisina/metabolismo , Camundongos , Chaperonas Moleculares/química , Células NIH 3T3 , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/química , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/química , Sumoilação
5.
Pediatr Res ; 70(2): 123-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21505375

RESUMO

Intrauterine growth restricted (IUGR) infants have increased susceptibility to infection associated with higher risk of illness and death. Dual specificity phosphatase 1 (DUSP1), which is transcribed in the thymus, increases in quantity as T cells mature and differentiate into CD4+ cells. Little is known about how IUGR affects DUSP1 levels and T-cell subpopulations over time. We hypothesized that IUGR would decrease cell count, CD4+ and CD8+ subpopulations of T lymphocytes, and DUSP1 levels in IUGR rat thymus and spleen. Bilateral uterine artery ligation produced IUGR rats. Thymus and spleen were harvested at P0 and P21. Flow cytometry was used to compare CD4+ and CD8+ lymphocyte populations. Real-time RT-PCR and Western blotting were used to determine DUSP1 quantity. IUGR significantly decreased total cell count in P0 and P21 IUGR male and female thymus. IUGR significantly increased CD4+ cells in IUGR P0 males and females, significantly decreased CD4+ cells in P21 female thymus, and significantly altered DUSP1 levels in the IUGR female thymus at P0 and P21, although it is not yet known whether the change in DUSP1 levels is due to a change in the level per cell or to a change in cellular composition of the thymus.


Assuntos
Diferenciação Celular/imunologia , Fosfatase 1 de Especificidade Dupla/metabolismo , Retardo do Crescimento Fetal/enzimologia , Retardo do Crescimento Fetal/imunologia , Linfócitos T/imunologia , Timo/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Western Blotting , Relação CD4-CD8 , Contagem de Células , Primers do DNA/genética , Feminino , Citometria de Fluxo , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
J Immunol ; 186(2): 697-707, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21148798

RESUMO

The reduced efficiency of the mammalian immune system with aging increases host susceptibility to infectious and autoimmune diseases. However, the mechanisms responsible for these pathologic changes are not well understood. In this study, we demonstrate that the bone marrow, blood, and secondary lymphoid organs of healthy aged mice possess increased numbers of immature myeloid cells that are phenotypically similar to myeloid-derived suppressor cells found in lymphoid organs of mice with progressive tumors and other pathologic conditions associated with chronic inflammation. These cells are characterized by the presence of Gr1 and CD11b markers on their surfaces. Gr1(+)CD11b(+) cells isolated from aged mice possess an ability to suppress T cell proliferation/activation and produce heightened levels of proinflammatory cytokines, both constitutively and upon activation, including IL-12, which promotes an excessive production of IFN-γ. IFN-γ priming is essential for excessive proinflammatory cytokine production and the suppressive activities by Gr1(+)CD11b(+) cells from aged mice. These cells suppress T cell proliferation through an NO-dependent mechanism, as depletion of splenic Gr1(+) cells reduces NO levels and restores T cell proliferation. Insights into mechanisms responsible for the proinflammatory and immune suppressive activities of Gr1(+)CD11b(+) cells from aged mice have uncovered a defective PI3K-Akt signaling pathway, leading to a reduced Akt-dependent inactivation of GSK3ß. Our data demonstrate that abnormal activities of the Gr1(+)CD11b(+) myeloid cell population from aged mice could play a significant role in the mechanisms responsible for immune senescence.


Assuntos
Envelhecimento/imunologia , Diferenciação Celular/imunologia , Células Mieloides/citologia , Células Mieloides/imunologia , Envelhecimento/genética , Animais , Antígeno CD11b/biossíntese , Antígeno CD11b/sangue , Antígeno CD11b/genética , Diferenciação Celular/genética , Proliferação de Células , Citocinas/biossíntese , Citocinas/fisiologia , Feminino , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Imunofenotipagem , Imunossupressão , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células Mieloides/metabolismo , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/sangue , Receptores de Quimiocinas/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia
7.
J Immunol ; 182(7): 4296-305, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19299729

RESUMO

The addition of monophosphoryl lipid A, a minimally toxic derivative of LPS, to nonmucosally administered vaccines induced both systemic and mucosal immune responses to coadministered Ags. This was dependent on an up-regulated expression of 1alpha-hydroxylase (CYP27B1, 1alphaOHase), the enzyme that converts 25-hydroxycholecalciferol, a circulating inactive metabolite of vitamin D(3), into 1,25(OH)2D(3) (calcitriol). In response to locally produced calcitriol, myeloid dendritic cells (DCs) migrated from cutaneous vaccination sites into multiple secondary lymphoid organs, including classical inductive sites of mucosal immunity, where they effectively stimulated B and T cell immune responses. The endogenous production of calcitriol by monophosphoryl lipid A-stimulated DCs appeared to be Toll-IL-1R domain-containing adapter-inducing IFN-beta-dependent, mediated through a type 1 IFN-induced expression of 1alphaOHase. Responsiveness to calcitriol was essential to promote the trafficking of mobilized DCs to nondraining lymphoid organs. Collectively, these studies help to expand our understanding of the physiologically important roles played by locally metabolized vitamin D(3) in the initiation and diversification of adaptive immune responses. The influences of locally produced calcitriol on the migration of activated DCs from sites of vaccination/infection into both draining and nondraining lymphoid organs create a condition whereby Ag-responsive B and T cells residing in multiple lymphoid organs are able to simultaneously engage in the induction of adaptive immune responses to peripherally administered Ags as if they were responding to an infection of peripheral or mucosal tissues they were designed to protect.


Assuntos
Adjuvantes Imunológicos/farmacologia , Colecalciferol/metabolismo , Células Dendríticas/imunologia , Receptores Toll-Like/imunologia , Vacinas/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Animais , Linfócitos B/imunologia , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Calcitriol/imunologia , Calcitriol/metabolismo , Movimento Celular/imunologia , Colecalciferol/imunologia , Células Dendríticas/metabolismo , Imunidade nas Mucosas/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Lipídeo A/farmacologia , Ativação Linfocitária/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Transgênicos , Receptores Toll-Like/metabolismo
8.
Infect Immun ; 76(11): 5191-9, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18765736

RESUMO

Immunity conferred by conventional vaccines is restricted to a narrow range of closely related strains, highlighting the unmet medical need for the development of vaccines that elicit protection against multiple pathogenic serotypes. Here we show that a Salmonella bivalent vaccine comprised of strains that lack and overproduce DNA adenine methylase (Dam) conferred cross-protective immunity to salmonella clinical isolates of human and animal origin. Protective immunity directly correlated with increased levels of cross-reactive opsonizing antibodies and memory T cells and a diminished expansion of myeloid-derived suppressor cells (MDSCs) that are responsible for the immune suppression linked to several conditions of host stress, including chronic microbial infections, traumatic insults, and many forms of cancer. Further, aged mice contained increased numbers of MDSCs and were more susceptible to Salmonella infection than young mice, suggesting a role for these cells in the immune declines associated with the natural aging process. These data suggest that interventions capable of reducing MDSC presence and activities may allow corresponding increases in B- and T-cell stimulation and benefit the ability of immunologically diverse populations to be effectively vaccinated as well as reducing the risk of susceptible individuals to infectious disease.


Assuntos
Anticorpos Antibacterianos/imunologia , Células Mieloides/imunologia , Salmonelose Animal/prevenção & controle , Vacinas contra Salmonella/imunologia , Salmonella/imunologia , Envelhecimento/imunologia , Animais , Reações Cruzadas , Células HeLa , Humanos , Memória Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia
9.
Vaccine ; 26(5): 601-13, 2008 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-18178294

RESUMO

Cathelicidin production by human myeloid cells stimulated through toll-like receptor (TLR) 2/1, the migration of human CD8+ T cells to inflamed skin sites, and the ability of murine dendritic cells (DCs) to migrate from skin sites of vaccination to mucosal lymphoid organs all occur via calcitriol-dependent mechanisms. Herein, we report that murine DCs exposed to TLR3/TLR4 ligands upregulate their expression of 1 alpha-hydroxylase, the enzyme that converts circulating 25(OH)D3 to calcitriol, the active form of vitamin D3. TLR3/TLR4 ligands injected subcutaneously affect DC migration in vivo, allowing their trafficking to both draining and non-draining systemic and mucosal lymphoid organs. Subcutaneously delivered vaccines containing TLR3/TLR4 ligands and antigen stimulate the induction of both systemic and mucosal immune responses. Vaccines containing TLR9 ligands fail to stimulate 1 alpha-hydroxylase protein expression, are incapable of redirecting DC migration into Peyer's patches and do not induce mucosal immune responses. These findings support a hypothesis that active metabolites of vitamin D3 produced locally are able to affect various aspects of innate and acquired immune responses.


Assuntos
Calcitriol/metabolismo , Colecalciferol/metabolismo , Células Dendríticas/fisiologia , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Vacinação , Animais , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Movimento Celular , Células Cultivadas , Hidrolases/metabolismo , Imunidade nas Mucosas , Injeções Subcutâneas , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos T/deficiência , Receptores de Antígenos de Linfócitos T/genética , Regulação para Cima
10.
Vaccine ; 25(7): 1236-49, 2007 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-17092617

RESUMO

The mucosal immune system provides the host with a first line of adaptive immune defense against invasion by many species of pathogenic microorganisms and their secreted products. Calcitriol, the active form of Vitamin D3 (VD3), promotes the induction of mucosal immunity in mice when added to subcutaneously administered vaccine formulations. Dendritic cells (DCs) activated at vaccination sites where VD3 is present gain the capacity to bypass sequestration in the draining lymph node and traffic to the Peyer's Patches (PP) of immunized animals. By employing protocols that allow the effective tracking of endogenous or adoptively transferred myeloid DCs in vivo, we found that VD3 influences on the trafficking of fully differentiated immature DCs were temporary, and occur without negative effects to antigen processing or peptide presentation to CD4+ T cells. In contrast, DCs differentiated from hematopoietic precursors in the presence of VD3 (conditioned DCs), were markedly compromised in their antigen presenting properties, while manifesting clear alterations to their trafficking properties in vivo. Similar to the recent finding of VD3-mediated enhancement of innate immune protection, our findings suggest that VD3 could also play an important role in controlling the types of immune effector responses elicited subsequent to either infection or vaccination.


Assuntos
Adjuvantes Imunológicos , Apresentação do Antígeno/efeitos dos fármacos , Apresentação do Antígeno/imunologia , Colecalciferol/farmacologia , Células Dendríticas/imunologia , Células Mieloides/imunologia , Animais , Células da Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Células Dendríticas/efeitos dos fármacos , Feminino , Imunidade nas Mucosas/imunologia , Imunoterapia Adotiva , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Células Mieloides/efeitos dos fármacos , Receptores CCR7 , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Pele/citologia , Pele/imunologia
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