RESUMO
The nucleosome remodeling and deacetylase (NuRD) complex modifies nucleosome positioning and chromatin compaction to regulate gene expression. The methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) play a critical role in complex formation; however, the molecular details of how they interact with other NuRD components have yet to be fully elucidated. We previously showed that an intrinsically disordered region (IDR) of MBD2 is necessary and sufficient to bind to the histone deacetylase core of NuRD. Building on that work, we have measured the inherent structural propensity of the MBD2-IDR using solvent and site-specific paramagnetic relaxation enhancement measurements. We then used the AlphaFold2 machine learning software to generate a model of the complex between MBD2 and the histone deacetylase core of NuRD. This model is remarkably consistent with our previous studies, including the current paramagnetic relaxation enhancement data. The latter suggests that the free MBD2-IDR samples conformations similar to the bound structure. We tested this model of the complex extensively by mutating key contact residues and measuring binding using an intracellular bioluminescent resonance energy transfer assay. Furthermore, we identified protein contacts that, when mutated, disrupted gene silencing by NuRD in a cell model of fetal hemoglobin regulation. Hence, this work provides insights into the formation of NuRD and highlights critical binding pockets that may be targeted to block gene silencing for therapy. Importantly, we show that AlphaFold2 can generate a credible model of a large complex that involves an IDR that folds upon binding.
Assuntos
Histona Desacetilases , Nucleossomos , Histona Desacetilases/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Inativação Gênica , Cromatina , Histona Desacetilase 1/genéticaRESUMO
During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2-NuRD. In this report, we present direct evidence that MBD2-NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a-NuRD, and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine-rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine-binding domain (MBD) of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings support a unified model that integrates the respective roles of BCL11A, MBD2a-NuRD, PRMT5, and DNA methylation in HbF silencing.
Assuntos
Hemoglobina Fetal , gama-Globinas , Adulto , Recém-Nascido , Humanos , Genes Reguladores , Fatores de Transcrição , Cromatina , Citosina , Proteína-Arginina N-Metiltransferases , Proteínas de Ligação a DNARESUMO
As high fetal hemoglobin levels ameliorate the underlying pathophysiological defects in sickle cell anemia and beta (ß)-thalassemia, understanding the mechanisms that enforce silencing of fetal hemoglobin postnatally offers the promise of effective molecular therapy. Depletion of the Nucleosome Remodeling and Deacetylase complex member MBD2 causes a 10-20-fold increase in γ-globin gene expression in adult ß-globin locus yeast artificial chromosome transgenic mice. To determine the effect of MBD2 depletion in human erythroid cells, genome editing technology was utilized to knockout MBD2 in Human Umbilical cord Derived Erythroid Progenitor-2 cells resulting in γ/γ+ß mRNA levels of approximately 50% and approximately 40% fetal hemoglobin by high performance liquid chromatography. In contrast, MBD3 knockout had no appreciable effect on γ-globin expression. Knockdown of MBD2 in primary adult erythroid cells consistently increased γ/γ+ß mRNA ratios by approximately 10-fold resulting in approximately 30-40% γ/γ+ß mRNA levels and a corresponding increase in γ-globin protein. MBD2 exerts its repressive effects through recruitment of the chromatin remodeler CHD4 via a coiled-coil domain, and the histone deacetylase core complex via an intrinsically disordered region. Enforced expression of wild-type MBD2 in MBD2 knockout cells caused a 5-fold decrease in γ-globin mRNA while neither the coiled-coil mutant nor the intrinsically disordered region mutant MBD2 proteins had an inhibitory effect. Co-immunoprecipitation assays showed that the coiled-coil and intrinsically disorder region mutations disrupt complex formation by dissociating the CHD4 and the histone deacetylase core complex components, respectively. These results establish the MBD2 Nucleosome Remodeling and Deacetylase complex as a major silencer of fetal hemoglobin in human erythroid cells and point to the coiled-coil and intrinsically disordered region of MBD2 as potential therapeutic targets.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Eritroides/metabolismo , Hemoglobina Fetal/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Mutação , gama-Globinas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adulto , Células Cultivadas , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Células Eritroides/citologia , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/antagonistas & inibidores , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double-stranded break (DSB) repair. Here, we show that depletion of CHD4 in acute myeloid leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ataxia-telangiectasia mutated pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic agent-induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34(+) hematopoietic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor-forming behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Antimetabólitos Antineoplásicos/uso terapêutico , Autoantígenos/genética , Citarabina/uso terapêutico , Daunorrubicina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Interferência de RNA , Células Tumorais CultivadasRESUMO
The MBD2-NuRD (Nucleosome Remodeling and Deacetylase) complex is an epigenetic reader of DNA methylation that regulates genes involved in normal development and neoplastic diseases. To delineate the architecture and functional interactions of the MBD2-NuRD complex, we previously solved the structures of MBD2 bound to methylated DNA and a coiled-coil interaction between MBD2 and p66α that recruits the CHD4 nucleosome remodeling protein to the complex. The work presented here identifies novel structural and functional features of a previously uncharacterized domain of MBD2 (MBD2IDR). Biophysical analyses show that the MBD2IDR is an intrinsically disordered region (IDR). However, despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical contact region requiring two contiguous amino acid residues, Arg(286) and Leu(287). Mutating these residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene PRSS8 in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Metilação de DNA , Proteínas de Ligação a DNA/genética , Epigênese Genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.
Assuntos
Proteínas Aviárias/química , Ilhas de CpG/fisiologia , Proteínas de Ligação a DNA/química , DNA/química , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas , DNA/genética , DNA/metabolismo , Metilação de DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de ProteínaRESUMO
An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate ß-type globin gene disorders such as sickle cell anemia and ß-thalassemia through activation of the fetal γ-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD, play a role in γ-globin gene silencing, and Mi2ß (CHD4) is a critical component of NuRD complexes. We observed that knockdown of Mi2ß relieves γ-globin gene silencing in ß-YAC transgenic murine chemical inducer of dimerization hematopoietic cells and in CD34(+) progenitor-derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2ß binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for γ-globin gene silencing during ß-type globin gene switching. Remarkably, <50% knockdown of Mi2ß is sufficient to significantly induce γ-globin gene expression without disrupting erythroid differentiation of primary human CD34(+) progenitors. These results indicate that Mi2ß is a potential target for therapeutic induction of fetal hemoglobin.
Assuntos
Autoantígenos/metabolismo , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Regulação da Expressão Gênica , Inativação Gênica , Células-Tronco Hematopoéticas/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , gama-Globinas/genética , Adulto , Animais , Autoantígenos/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Células Eritroides/citologia , Hemoglobina Fetal/antagonistas & inibidores , Hemoglobina Fetal/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , Proteínas Repressoras , gama-Globinas/antagonistas & inibidores , gama-Globinas/metabolismoRESUMO
Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal ß-type globin gene silencing during development in vivo. Here we present structural and biophysical details of the coiled-coil interaction between MBD2 and p66α, a critical component of the MBD2-NuRD complex. We show that enforced expression of the isolated p66α coiled-coil domain relieves MBD2-mediated globin gene silencing and that the expressed peptide interacts only with a subset of components of the MBD2-NuRD complex that does not include native p66α or Mi-2. These results demonstrate the central importance of the coiled-coil interaction and suggest that MBD2-dependent DNA methylation-driven gene silencing can be disrupted by selectively targeting this coiled-coil complex.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Modelos Moleculares , Proteínas Repressoras/metabolismo , Western Blotting , Metilação de DNA/genética , Primers do DNA/genética , Inativação Gênica , Humanos , Imunoprecipitação , Interferência de RNARESUMO
The epigenetic code of DNA methylation is interpreted chiefly by methyl cytosine binding domain (MBD) proteins which in turn recruit multiprotein co-repressor complexes. We previously isolated one such complex, MBD2-NuRD, from primary erythroid cells and have shown it contributes to embryonic/fetal ß-type globin gene silencing during development. This complex has been implicated in silencing tumor suppressor genes in a variety of human tumor cell types. Here we present structural details of chicken MBD2 bound to a methylated DNA sequence from the ρ-globin promoter to which it binds in vivo and mediates developmental transcriptional silencing in normal erythroid cells. While previous studies have failed to show sequence specificity for MBD2 outside of the symmetric mCpG, we find that this domain binds in a single orientation on the ρ-globin target DNA sequence. Further, we show that the orientation and affinity depends on guanine immediately following the mCpG dinucleotide. Dynamic analyses show that DNA binding stabilizes the central ß-sheet, while the N- and C-terminal regions of the protein maintain mobility. Taken together, these data lead to a model in which DNA binding stabilizes the MBD2 structure and that binding orientation and affinity is influenced by the DNA sequence surrounding the central mCpG.
Assuntos
Proteínas Aviárias/química , Metilação de DNA , Proteínas de Ligação a DNA/química , DNA/química , Animais , Sequência de Bases , Galinhas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: African American race and uninsurance are associated with undertreatment and poor survival in solid tumor cancers. This relationship has not been examined in acute myeloid leukemia (AML) where absence of treatment or treatment delays can result in death within weeks or months. Induction followed by consolidation treatment, in contrast, has a high probability for remission or cure. We examined the relationship between race and health insurance and inpatient chemotherapy and survival in AML patients between the ages of 21 and 64 years. We also examined inpatient costs associated with inpatient treatment. METHODS: We used population-based data from the Virginia Cancer Registry and the Virginia Health Information discharge data for patients diagnosed with AML between 1999 and 2006 (n = 523). Adjusted logistic regression was used to measure the relationship between the independent variables and chemotherapy. We used the Cox proportional hazards method to estimate survival. RESULTS: Uninsured patients were more likely to be untreated than their privately insured counterparts (odds ratio, 4.40; 95% confidence interval, 1.85-10.49) and had a higher likelihood of death (hazard ratio, 1.29; 95% confidence interval, 1.02-1.84). Once treatment was adjusted in the survival analyses, differences between insurance groups were not statistically significant. The median 1-year cost of inpatient care following diagnosis for patients who received chemotherapy exceeded $100,000. CONCLUSION: This study addressed the urgency for health insurance that affords access to care. Without treatment, the outcome of AML is death within only a few months; with treatment, the chance for long-term remission or even cure exists.
Assuntos
Antineoplásicos/economia , Negro ou Afro-Americano/estatística & dados numéricos , Custos de Cuidados de Saúde , Acessibilidade aos Serviços de Saúde/economia , Seguro Saúde , Leucemia Mieloide Aguda , Pessoas sem Cobertura de Seguro de Saúde/estatística & dados numéricos , População Branca/estatística & dados numéricos , Adulto , Antineoplásicos/uso terapêutico , Transplante de Medula Óssea/economia , Fatores de Confusão Epidemiológicos , Etnicidade/estatística & dados numéricos , Feminino , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , Custos Hospitalares , Humanos , Pacientes Internados , Tempo de Internação/economia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/economia , Leucemia Mieloide Aguda/mortalidade , Modelos Logísticos , Masculino , Medicaid , Medicare , Pessoa de Meia-Idade , Razão de Chances , Modelos de Riscos Proporcionais , Sistema de Registros , Retratamento/economia , Estados Unidos , Virginia/epidemiologiaRESUMO
During erythroid development, the embryonic ε-globin gene becomes silenced as erythropoiesis shifts from the yolk sac to the fetal liver where γ-globin gene expression predominates. Previous studies have shown that the ε-globin gene is autonomously silenced through promoter proximal cis-acting sequences in adult erythroid cells. We have shown a role for the methylcytosine binding domain protein 2 (MBD2) in the developmental silencing of the avian embryonic ρ-globin and human fetal γ-globin genes. To determine the roles of MBD2 and DNA methylation in human ε-globin gene silencing, transgenic mice containing all sequences extending from the 5' hypersensitive site 5 (HS5) of the ß-globin locus LCR to the human γ-globin gene promoter were generated. These mice show correct developmental expression and autonomous silencing of the transgene. Either the absence of MBD2 or treatment with the DNA methyltransferase inhibitor 5-azacytidine increases ε-globin transgene expression by 15-20 fold in adult mice. Adult mice containing the entire human ß-globin locus also show an increase in expression of both the ε-globin gene transgene and endogenous ε(Y) and ß(H1) genes in the absence of MBD2. These results indicate that the human ε-globin gene is subject to multilayered silencing mediated in part by MBD2.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Globinas épsilon/genética , Animais , Azacitidina/farmacologia , Metilação de DNA , Eritroblastos/metabolismo , Eritrócitos/metabolismo , Feminino , Ordem dos Genes , Hemoglobinas Anormais/metabolismo , Humanos , Região de Controle de Locus Gênico/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Globinas beta/metabolismo , gama-Globinas/metabolismoRESUMO
DNA methylation represents a fundamental epigenetic modification that regulates chromatin architecture and gene transcription. Many diseases, including cancer, show aberrant methylation patterns that contribute to the disease phenotype. DNA methylation inhibitors have been used to block methylation dependent gene silencing to treat hematopoietic neoplasms and to restore expression of developmentally silenced genes. However, these inhibitors disrupt methylation globally and show significant off-target toxicities. As an alternative approach, we have been studying readers of DNA methylation, the 5-methylcytosine binding domain family of proteins, as potential therapeutic targets to restore expression of aberrantly and developmentally methylated and silenced genes. In this review, we discuss the role of DNA methylation in gene regulation and cancer development, the structure and function of the 5-methylcytosine binding domain family of proteins, and the possibility of targeting the complexes these proteins form to treat human disease.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Animais , Metilação de DNA/efeitos dos fármacos , Humanos , Modelos MolecularesRESUMO
Human umbilical cord blood is a rich source of hematopoietic stem and progenitor cells. CD34+ cells in umbilical cord blood are more primitive than those in peripheral blood or bone marrow, and can proliferate at a high rate and differentiate into multiple cell types. In this protocol, a dependable method is described for the isolation of fetal CD34+ cells from umbilical cord blood and expanding these cells in culture. The cells can then be in vitro differentiated along an erythroid pathway, while simultaneously performing knockdown of a gene of choice. The use of lentiviral vectors that express small hairpin RNA (shRNA) is an efficient method to downregulate genes. Flow cytometric analyses are used to enrich for erythroid cells. Using these methods, one can generate in vitro differentiated cells to use for quantitative reverse transcriptase PCR and other purposes.
Assuntos
Diferenciação Celular/genética , Sangue Fetal/citologia , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Antígenos CD34/metabolismo , Técnicas de Cultura de Células , Separação Celular , Células Eritroides/citologia , Células Eritroides/metabolismo , Citometria de Fluxo , Expressão Gênica , Células HEK293 , Humanos , TransfecçãoRESUMO
The human major histocompatibility complex (MHC) class Ib gene, HLA-E, codes for the major ligand of the inhibitory receptor NK-G-2A, which is present on most natural killer (NK) cells and some CD8(+) cytotoxic T lymphocytes. We have previously shown that gamma interferon (IFN-gamma) induction of HLA-E gene transcription is mediated through a distinct IFN-gamma-responsive element, the IFN response region (IRR), in all cell types studied. We have now identified and characterized a cell type-restricted enhancer of IFN-gamma-mediated induction of HLA-E gene transcription, designated the upstream interferon response region (UIRR), which is located immediately upstream of the IRR. The UIRR mediates a three- to eightfold enhancement of IFN-gamma induction of HLA-E transcription in some cell lines but not in others, and it functions only in the presence of an adjacent IRR. The UIRR contains a variant GATA binding site (AGATAC) that is critical to both IFN-gamma responsiveness and to the formation of a specific binding complex containing GATA-1 in K562 cell nuclear extracts. The binding of GATA-1 to this site in response to IFN-gamma was confirmed in vivo in a chromatin immunoprecipitation assay. Forced expression of GATA-1 in nonexpressing U937 cells resulted in a four- to fivefold enhancement of the IFN-gamma response from HLA-E promoter constructs containing a wild-type but not a GATA-1 mutant UIRR sequence and increased the IFN-gamma response of the endogenous HLA-E gene. Knockdown of GATA-1 expression in K562 cells resulted in a approximately 4-fold decrease in the IFN-gamma response of the endogenous HLA-E gene, consistent with loss of the increase in IFN-gamma response of HLA-E promoter-driven constructs containing the UIRR in wild-type K562 cells. Coexpression of wild-type and mutant adenovirus E1a proteins that sequester p300/CBP eliminated IFN-gamma-mediated enhancement through the UIRR, but only partially reduced induction through the IRR, implicating p300/CBP binding to Stat-1alpha at the IRR in the recruitment of GATA-1 to mediate the cooperation between the UIRR and IRR. We propose that the GATA-1 transcription factor represents a cell type-restricted mediator of IFN-gamma induction of the HLA-E gene.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Antígenos HLA/genética , Antígenos de Histocompatibilidade Classe I/genética , Interferon gama/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Bases , Linhagem Celular , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Genes Reporter , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Elementos de Resposta , Fatores de Transcrição/genética , Transfecção , Antígenos HLA-ERESUMO
The developmental regulation of globin gene expression has served as an important model for understanding higher eukaryotic transcriptional control mechanisms. During human erythroid development, there is a sequential switch from expression of the embryonic ε-globin gene to the fetal É£-globin gene in utero, and postpartum the É£-globin gene is silenced, as the ß-globin gene becomes the predominantly expressed locus. Because the expression of normally silenced fetal É£-type globin genes and resultant production of fetal hemoglobin (HbF) in adult erythroid cells can ameliorate the pathophysiological consequences of both abnormal ß-globin chains in sickle cell anemia and deficient ß-globin chain production in ß-thalassemia, understanding the complex mechanisms of this developmental switch has direct translational clinical relevance. Of particular interest for translational research are the factors that mediate silencing of the É£-globin gene in adult stage erythroid cells. In addition to the regulatory roles of transcription factors and their cognate DNA sequence motifs, there has been a growing appreciation of the role of epigenetic signals and their cognate factors in gene regulation, and in particular in gene silencing through chromatin. Much of the information about epigenetic silencing stems from studies of globin gene regulation. As discussed here, the term epigenetics refers to postsynthetic modifications of DNA and chromosomal histone proteins that affect gene expression and can be inherited through somatic cell replication. A full understanding of the molecular mechanisms of epigenetic silencing of HbF expression should facilitate the development of more effective treatment of ß-globin chain hemoglobinopathies.
Assuntos
Epigênese Genética , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Animais , Ensaios Clínicos como Assunto , Metilação de DNA , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Hemoglobinopatias/genética , Histonas/metabolismo , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismo , Pesquisa Translacional Biomédica , Globinas beta/genética , Talassemia beta/genética , gama-Globinas/genéticaRESUMO
Methyl cytosine binding domain protein 2 (MBD2) has been shown to bind to and mediate repression of methylated tumor suppressor genes in cancer cells, where repatterning of CpG methylation and associated gene silencing is common. We have investigated the role of MBD2 in breast cancer cell growth and tumor suppressor gene expression. We show that stable short hairpin RNA (shRNA)-mediated knockdown of MBD2 leads to growth suppression of cultured human mammary epithelial cancer lines, SK-BR-3, MDA-MB-231, and MDA-MB-435. The peak antiproliferative occurs only after sustained, stable MBD2 knockdown. Once established, the growth inhibition persists over time and leads to a markedly decreased propensity for aggressive breast cancer cell lines to form in vivo xenograft tumors in Bagg Albino (BALB)/C nu/nu mice. The growth effects of MBD2 knockdown are accompanied by derepression of tumor suppressor genes, including DAPK1 and KLK10. Chromatin immunoprecipitation assays and bisulfite sequencing show MBD2 binding directly to the hyper methylated and CpG-rich promoters of both DAPK1 and KLK10. Remarkably, the promoter CpG island-associated methylation of these genes remained stable despite robust transcriptional activation in MBD2 knockdown cells. Expression of a shRNA-resistant MBD2 protein resulted in restoration of growth and resilencing of the MBD2-dependent tumor suppressor genes. Our data suggest that uncoupling CpG methylation from repressive chromatin remodeling and histone modifications by removing MBD2 is sufficient to initiate and maintain tumor suppressor gene transcription and suppress neoplastic cell growth. These results show a role for MBD2 in cancer progression and provide support for the prospect of targeting MBD2 therapeutically in aggressive breast cancers.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Ilhas de CpG/genética , Metilação de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Estadiamento de Neoplasias , RNA Interferente Pequeno , Células Tumorais CultivadasRESUMO
The sequence complexity of the known vertebrate genomes alone is insufficient to account for the diversity between individuals of a species. Although our knowledge of vertebrate biology has evolved substantially with the growing compilation of sequenced genomes, understanding the temporal and spatial regulation of genes remains fundamental to fully exploiting this information. The importance of epigenetic factors in gene regulation was first hypothesized decades ago when biologists posited that methylation of DNA could heritably alter gene expression [Holliday and Pugh, 1975. Science 187(4173), 226-232; Riggs, 1975. Cytogenet. and Cell Genet.14(1), 9-25; Scarano et al., 1967. Proc. Natl. Acad. Sci. USA 57(5), 1394-1400)]. It was subsequently shown that vertebrate DNA methylation, almost exclusively at the 5' position of cytosine in the dinucleotide CpG, played a role in a number of processes including embryonic development, genetic imprinting, cell differentiation, and tumorigenesis. At the time of this writing, a large and growing list of genes is known to exhibit DNA methylation-dependent regulation, and we understand in some detail the mechanisms employed by cells in using methylation as a regulatory modality. In this context, we revisit one of the original systems in which the role of DNA methylation in vertebrate gene regulation during development was described and studied: erythroid cells. We briefly review the recent advances in our understanding of DNA methylation and, in particular, its regulatory role in red blood cells during differentiation and development. We also address DNA methylation as a component of erythroid chromatin architecture, and the interdependence of CpG methylation and histone modification.
Assuntos
Metilação de DNA , Epigênese Genética/genética , Células Eritroides/citologia , Células Eritroides/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Globinas/genética , HumanosRESUMO
The genes of the vertebrate beta-globin locus undergo a switch in expression during erythroid development whereby embryonic/fetal genes of the cluster are sequentially silenced and adult genes are activated. We describe here a role for DNA methylation and MBD2 in the silencing of the human fetal gamma-globin gene. The gamma-globin gene is reactivated upon treatment with the DNA methyltransferase inhibitor 5-azacytidine in the context of a mouse containing the entire human beta-globin locus as a yeast artificial chromosome (betaYAC) transgene. To elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2-/- mice were generated by breeding betaYAC mice with MBD2-/- mice. Adult betaYAC/MBD2-/- mice continue to express the gamma-globin gene at a level commensurate with 5-azacytidine treatment, 10- to 20-fold over that observed with 1-acetyl-2-phenylhydrazine treatment alone. In addition, the level of gamma-globin expression is consistently higher in MBD2-/- mice in 14.5- and 16.5-days postcoitus fetal liver erythroblasts suggesting a role for MBD2 in embryonic/fetal erythroid development. DNA methylation levels are modestly decreased in MBD2-/- mice. MBD2 does not bind to the gamma-globin promoter region to maintain gamma-globin silencing. Finally, treatment of MBD2-null mice with 5-azacytidine induces only a small, nonadditive induction of gamma-globin mRNA, signifying that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells.