RESUMO
The tumor suppressor IKAROS binds and represses multiple NOTCH target genes. For their induction upon NOTCH signaling, IKAROS is removed and replaced by NOTCH Intracellular Domain (NICD)-associated proteins. However, IKAROS remains associated to other NOTCH activated genes upon signaling and induction. Whether IKAROS could participate to the induction of this second group of NOTCH activated genes is unknown. We analyzed the combined effect of IKAROS abrogation and NOTCH signaling on the expression of NOTCH activated genes in erythroid cells. In IKAROS-deleted cells, we observed that many of these genes were either overexpressed or no longer responsive to NOTCH signaling. IKAROS is then required for the organization of bivalent chromatin and poised transcription of NOTCH activated genes belonging to either of the aforementioned groups. Furthermore, we show that IKAROS-dependent poised organization of the NOTCH target Cdkn1a is also required for its adequate induction upon genotoxic insults. These results highlight the critical role played by IKAROS in establishing bivalent chromatin and transcriptional poised state at target genes for their activation by NOTCH or other stress signals.
Assuntos
Regulação da Expressão Gênica , Fator de Transcrição Ikaros/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Sequenciamento de Cromatina por Imunoprecipitação , Feminino , Fator de Transcrição Ikaros/genética , Masculino , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Ligação Proteica , RNA Interferente PequenoRESUMO
Transcription factors are important determinants of lineage specification during hematopoiesis. They favor recruitment of cofactors involved in epigenetic regulation, thereby defining patterns of gene expression in a development- and lineage-specific manner. Additionally, transcription factors can facilitate transcription preinitiation complex (PIC) formation and assembly on chromatin. Interestingly, a few lineage-specific transcription factors, including IKAROS, also regulate transcription elongation. IKAROS is a tumor suppressor frequently inactivated in leukemia and associated with a poor prognosis. It forms a complex with the nucleosome remodeling and deacetylase (NuRD) complex and the positive transcription elongation factor b (P-TEFb), which is required for productive transcription elongation. It has also been reported that IKAROS interacts with factors involved in transcription termination. Here we review these and other recent findings that establish IKAROS as the first transcription factor found to act as a multifunctional regulator of the transcription cycle in hematopoietic cells.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Polimerase II/fisiologia , Fator de Transcrição Ikaros/fisiologia , Transcrição Gênica , Animais , Regulação da Expressão Gênica , HumanosRESUMO
IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of Ik(NULL) hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.
Assuntos
Montagem e Desmontagem da Cromatina , Hematopoese , Fator de Transcrição Ikaros/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Animais , Células COS , Chlorocebus aethiops , Montagem e Desmontagem da Cromatina/genética , Hematopoese/genética , Humanos , Fator de Transcrição Ikaros/genética , Células Jurkat , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Camundongos Knockout , Nucleossomos/metabolismo , Ligação Proteica , RNA Polimerase II/metabolismo , Ativação TranscricionalRESUMO
Inflammation control is critical during the innate immune response. Such response is triggered by the detection of molecules originating from pathogens or damaged host cells by pattern-recognition receptors (PRRs). PRRs subsequently initiate intra-cellular signalling through different pathways, resulting in i) the production of inflammatory cytokines, including type I interferon (IFN), and ii) the initiation of a cascade of events that promote both immediate host responses as well as adaptive immune responses. All human PYRIN and HIN-200 domains (PYHIN) protein family members were initially proposed to be PRRs, although this view has been challenged by reports that revealed their impact on other cellular mechanisms. Of relevance here, the human PYHIN factor myeloid nuclear differentiation antigen (MNDA) has recently been shown to directly control the transcription of genes encoding factors that regulate programmed cell death and inflammation. While MNDA is mainly found in the nucleus of leukocytes of both myeloid (neutrophils and monocytes) and lymphoid (B-cell) origin, its subcellular localization has been shown to be modulated in response to genotoxic agents that induce apoptosis and by bacterial constituents, mediators of inflammation. Prior studies have noted the importance of MNDA as a marker for certain forms of lymphoma, and as a clinical prognostic factor for hematopoietic diseases characterized by defective regulation of apoptosis. Abnormal expression of MNDA has also been associated with altered levels of cytokines and other inflammatory mediators. Refining our comprehension of the regulatory mechanisms governing the expression of MNDA and other PYHIN proteins, as well as enhancing our definition of their molecular functions, could significantly influence the management and treatment strategies of numerous human diseases. Here, we review the current state of knowledge regarding PYHIN proteins and their role in innate and adaptive immune responses. Emphasis will be placed on the regulation, function, and relevance of MNDA expression in the control of gene transcription and RNA stability during cell death and inflammation.
Assuntos
Antígenos de Diferenciação Mielomonocítica , Apoptose , Regulação da Expressão Gênica , Fatores de Transcrição , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Animais , Imunidade Inata , Transcrição Gênica , Inflamação/imunologia , Transdução de SinaisRESUMO
Ikaros is associated with both gene transcriptional activation and repression in lymphocytes. Ikaros acts also as repressor of human γ-globin (huγ-) gene transcription in fetal and adult erythroid cells. Whether and eventually, how Ikaros can function as a transcriptional activator in erythroid cells remains poorly understood. Results presented herein demonstrate that Ikaros is a developmental-specific activator of huγ-gene expression in yolk sac erythroid cells. Molecular analysis in primary cells revealed that Ikaros interacts with Gata-1 and favors Brg1 recruitment to the human ß-globin Locus Control Region and the huγ-promoters, supporting long-range chromatin interactions between these regions. Additionally, we demonstrate that Ikaros contributes to transcription initiation and elongation of the huγ-genes, since it is not only required for TBP and RNA Polymerase II (Pol II) assembly at the huγ-promoters but also for conversion of Pol II into the elongation-competent phosphorylated form. In agreement with the latter, we show that Ikaros interacts with Cyclin-dependent kinase 9 (Cdk9), which contributes to efficient transcription elongation by phosphorylating the C-terminal domain of the large subunit of Pol II on Serine 2, and favours Cdk9 recruitment to huγ-promoters. Our results show that Ikaros exerts dual functionality during gene activation, by promoting efficient transcription initiation and elongation.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Células Eritroides/metabolismo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição Ikaros/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , DNA Helicases/metabolismo , Humanos , Fator de Transcrição Ikaros/genética , Região de Controle de Locus Gênico , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Saco Vitelino/anatomia & histologia , Saco Vitelino/crescimento & desenvolvimento , gama-Globulinas/genética , gama-Globulinas/metabolismoRESUMO
IKAROS is a master regulator of cell fate determination in lymphoid and other hematopoietic cells. This transcription factor orchestrates the association of epigenetic regulators with chromatin, ensuring the expression pattern of target genes in a developmental and lineage-specific manner. Disruption of IKAROS function has been associated with the development of acute lymphocytic leukemia, lymphoma, chronic myeloid leukemia and immune disorders. Paradoxically, while IKAROS has been shown to be a tumor suppressor, it has also been identified as a key therapeutic target in the treatment of various forms of hematological malignancies, including multiple myeloma. Indeed, targeted proteolysis of IKAROS is associated with decreased proliferation and increased death of malignant cells. Although the molecular mechanisms have not been elucidated, the expression levels of IKAROS are variable during hematopoiesis and could therefore be a key determinant in explaining how its absence can have seemingly opposite effects. Mechanistically, IKAROS collaborates with a variety of proteins and complexes controlling chromatin organization at gene regulatory regions, including the Nucleosome Remodeling and Deacetylase complex, and may facilitate transcriptional repression or activation of specific genes. Several transcriptional regulatory functions of IKAROS have been proposed. An emerging mechanism of action involves the ability of IKAROS to promote gene repression or activation through its interaction with the RNA polymerase II machinery, which influences pausing and productive transcription at specific genes. This control appears to be influenced by IKAROS expression levels and isoform production. In here, we summarize the current state of knowledge about the biological roles and mechanisms by which IKAROS regulates gene expression. We highlight the dynamic regulation of this factor by post-translational modifications. Finally, potential avenues to explain how IKAROS destruction may be favorable in the treatment of certain hematological malignancies are also explored.
RESUMO
Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human beta-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the beta-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual beta-like globin genes. Moreover, in transgenic mice expressing the entire human beta-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter beta-like globin gene expression. Here, we show that abnormal expression of human beta-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human epsilon- and gamma-promoters, but not at the beta-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human gamma-promoters. We also find that HS2 is dispensable for high-level human beta-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement.
Assuntos
Globinas/genética , Região de Controle de Locus Gênico , Regiões Promotoras Genéticas , Animais , Cromatina/química , Cromatina/metabolismo , Células Eritroides/metabolismo , Expressão Gênica , Globinas/metabolismo , Fígado/embriologia , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Deleção de Sequência , Fatores de Transcrição/metabolismo , Transcrição Gênica , Saco Vitelino/citologiaRESUMO
The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course.
Assuntos
Antígenos de Diferenciação Mielomonocítica/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Fatores de Transcrição/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígenos de Diferenciação Mielomonocítica/genética , Apoptose/genética , Cromatina/genética , Cromatina/metabolismo , Feminino , Células HL-60 , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fatores de Transcrição/genéticaRESUMO
Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA3/metabolismo , Hematopoese , Fator de Transcrição Ikaros/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Células Cultivadas , Quinase 9 Dependente de Ciclina/química , Fator de Transcrição GATA1/química , Fator de Transcrição GATA2/química , Fator de Transcrição GATA3/química , Fator de Transcrição Ikaros/química , Camundongos , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human beta-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human gamma-gene promoters, thereby contributing to gamma-globin gene silencing at the time of the gamma- to beta-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human gamma-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.
Assuntos
Fator de Transcrição GATA1/fisiologia , Inativação Gênica , Fator de Transcrição Ikaros/fisiologia , gama-Globinas/metabolismo , Animais , Eritropoese/genética , Regulação da Expressão Gênica , Histona Desacetilase 1 , Histona Desacetilases/metabolismo , Humanos , Fator de Transcrição Ikaros/genética , Técnicas In Vitro , Células K562 , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , gama-Globinas/genéticaRESUMO
Basal expression of lineage-specific transcription factors (TFs) in multipotent hematopoietic progenitor cells (HPCs) plays a pivotal role in normal hematopoiesis. Indeed, the interplay between lineage-specific TFs and chromatin modifying or remodeling complexes allows chromatin modifications at specific hematopoietic loci and promotes transcriptionally prone conformations. During hematopoiesis, the expression of various lineage-specific genes can be preceded by their potentiation i.e., by chromatin activation, in progenitor cells. Gene potentiation appears to counterbalance epigenetic silencing of lineage-specific genes in early progenitors, while maintaining an accessible chromatin conformation in the lineage pathway selected. Herein, we discuss the impact of lineage-specific TFs on gene potentiation and priming in normal hematopoiesis, and emphasize the complementary role of locus control region (LCR) or LCR-like structures and promoter regions in gene-specific potentiation events.
Assuntos
Linhagem da Célula , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Globinas/genética , Globinas/metabolismo , Hematopoese/fisiologia , Humanos , Região de Controle de Locus Gênico , Modelos Biológicos , Regiões Promotoras GenéticasRESUMO
During development, the regulated expression of tissue-specific genes can be preceded by their potentiation, that is, by chromatin activation in progenitor cells. For example, the human beta-like globin genes are potentiated in a gene- and developmental-specific manner in hematopoietic progenitors. Developmental regulation of human beta-gene expression in erythroid cells is mostly determined by transcriptional activators; however, it is not clear how gene-specific potentiation is set in hematopoietic progenitors. Using human and transgenic multipotent hematopoietic progenitors, we demonstrate that human beta-globin locus activation is characterized by TBP, NF-E2, CBP and BRG1 recruitment at both the Locus Control Region and human beta-gene promoter. Our results further indicate that in hematopoietic progenitors, EKLF influences chromatin organization at the human beta-globin locus and is instrumental for human beta-gene potentiation. Thus, we show that lineage-specific transcriptional activators expressed at basal levels in progenitor cells can participate in gene potentiation.
Assuntos
Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Globinas/genética , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Ativação Transcricional , Animais , Linhagem da Célula/fisiologia , Cromatina/metabolismo , DNA Helicases , Feminino , Globinas/metabolismo , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Subunidade p45 do Fator de Transcrição NF-E2/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismoRESUMO
Abnormal epigenetic regulation of gene expression contributes significantly to a variety of human pathologies including cancer. Deletion of hypersensitive site 2 (HS2) at the human beta-globin locus control region can lead to abnormal epigenetic regulation of globin genes in transgenic mice. Here, two HS2-deleted transgenic mouse lines were used as model to demonstrate that heritable alteration of chromatin organization at the human beta-globin locus in multipotent hematopoietic progenitors contributes to the abnormal expression of the beta-globin gene in mature erythroid cells. This alteration is characterized by specific patterns of histone covalent modifications that are inherited during erythropoiesis and, moreover, is plastic because it can be reverted by transient treatment with the histone deacetylase inhibitor Trichostatin A. Altogether, our results indicate that aberrant epigenetic regulation can be detected and modified before tissue-specific gene transcription, a finding which may lead to novel strategies for the prevention of chromatin-related pathologies.
Assuntos
Células da Medula Óssea/fisiologia , Cromatina/metabolismo , Eritropoese/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Globinas/genética , Região de Controle de Locus Gênico , Acetilação , Animais , Células da Medula Óssea/citologia , Cromatina/patologia , Elementos Facilitadores Genéticos/genética , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Transgênicos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
To study epigenetic regulation of the human beta-globin locus during hematopoiesis, we investigated patterns of histone modification and chromatin accessibility along this locus in hematopoietic progenitor cells (HPCs) derived from both humans and transgenic mice. We demonstrate that the developmentally related activation of human beta-like globin genes in humans and transgenic mice HPCs is preceded by a wave of gene-specific histone H3 hyperacetylation and K4 dimethylation. In erythroid cells, expression of beta-like globin genes is associated with histone hyperacetylation along these genes and, surprisingly, with local deacetylation at active promoters. We also show that endogenous mouse beta major and human beta-like genes are subject to different epigenetic control mechanisms in HPCs. This difference is likely due to intrinsic properties of the human beta-globin locus since, in transgenic mice, this locus is epigenetically regulated in the same manner as in human HPCs. Our results suggest that a defined pattern of histone H3 acetylation/dimethylation is important for specific activation of human globin promoters during development in human and transgenic HPCs. We propose that this transient acetylation/dimethylation is involved in gene-specific potentiation in HPCs (ie, before extensive chromatin remodeling and transcription take place in erythroid cells).