RESUMO
As epigenetic regulators are frequently dysregulated in acute myeloid leukemia (AML) we determined expression levels of the JmjC-protein NO66 in AML cell lines and sub fractions of healthy human hematopoietic cells. NO66 is absent in the AML cell lines KG1/KG1a which consist of cells with the immature CD34+/CD38- phenotype and is regarded as a "stem cell-like" model system. Similarly, NO66 is not detectable in CD34+/CD38- cells purified from healthy donors but is clearly expressed in the more committed CD34+/CD38+ cell population. Loss of NO66 expression in KG1/KG1a cells is due to hyper-methylation of its promoter and is released by DNA-methyltransferase inhibitors. In KG1a cells stably expressing exogenous wild type (KG1a66wt) or enzymatically inactive mutant (KG1a66mut) NO66, respectively, the wild type protein inhibited proliferation and rDNA transcription. Gene expression profiling revealed that the expression of NO66 induces a transcriptional program enriched for genes with roles in proliferation and maturation (e.g.EPDR1, FCER1A, CD247, MYCN, SNORD13). Genes important for the maintenance of stem cell properties are downregulated (e.g. SIRPA, Lin28B, JAML). Our results indicate that NO66 induces lineage commitment towards myeloid progenitor cell fate and suggest that NO66 contributes to loss of stem cell properties.
Assuntos
Proliferação de Células/genética , Dioxigenases/genética , Histona Desmetilases/genética , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Domínios Proteicos/genéticaRESUMO
Given the prominence and the biological importance of the nucleus it is remarkable how little is still known about structure-forming proteins in the nuclear interior. The karyoskeletal protein NO145 has been identified as a major constituent of a filamentous network surrounding the amplified nucleoli of Xenopus laevis oocytes. We now show that an orthologous protein also occurs in female germ cells of a wide range of other vertebrates, where it forms dot-like structures. Using the Xenopus oocyte system we further report a specific regulatory mechanism responsible for (1) the rapid degradation of the NO145 protein during meiotic maturation, and (2) the cell-type-dependent translation of NO145 mRNA. Microinjection experiments have revealed that NO145 is a target of proteasomes and the use of the rapid amplification of cDNA ends-polyadenylation test (RACE-PAT) has disclosed the existence of NO145 mRNAs differing in their 3' UTRs. Reporter systems as well as polyribosome profiling experiments have revealed the regulatory importance of the 3' UTRs, which affect the translational efficiency as well as the stability of the encoded protein. The highly conserved cell-type specificity and the extremely tight temporal regulation of NO145 synthesis suggest an important role of this protein in female meiotic prophase.
Assuntos
Proteínas do Citoesqueleto/biossíntese , Oócitos/metabolismo , Proteínas de Xenopus/biossíntese , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Primers do DNA , Genes Reporter , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
The nucleolus is the most prominent intranuclear structure of almost all protein-synthesizing cells. It compromises a well-defined functional compartmentalization and a high complexity of molecular constituents. Here, we report on the identification and molecular characterization of a novel constitutive nucleolar component--protein NO52--that is present in diverse species from Xenopus laevis to human. The cDNA-deduced amino acid sequence of protein NO52 defines a polypeptide of a calculated mass of 52.8 kDa and an isoelectric point of 6.7. Inspection of the primary sequence disclosed that the protein contains a JmjC domain and is highly sequence-related to the recently described nucleolar protein NO66. Immunolocalization studies revealed that protein NO52 is highly concentrated in the granular component of nucleoli and this characteristic intranuclear distribution is significantly affected by treatment of cells with (i) RNase A, (ii) actinomycin D and (iii) serum starvation. Interestingly, protein NO52 has been identified as a constituent of free preribosomal particles but is absent from cytoplasmic ribosomes. Analyses of immunocomplexes isolated from cellular extracts with an NO52-specific antibody by MALDI mass spectrometry further confirmed the interaction of protein NO52 with various ribosomal proteins as well as with a distinct set of non-ribosomal nucleolar proteins. The dependence of the nucleolar accumulation of the protein on ongoing rRNA transcription and the cellular metabolic state strongly suggest that protein NO52 is directly involved in ribosome biogenesis, most likely during the assembly process of preribosomal particles.
Assuntos
Nucléolo Celular/fisiologia , Proteínas Cromossômicas não Histona/genética , Sequência de Aminoácidos , Animais , Proteínas Cromossômicas não Histona/fisiologia , Dioxigenases , Histona Desmetilases , Humanos , Imuno-Histoquímica , Dados de Sequência MolecularRESUMO
Polycomb group (PcG) proteins are responsible for the stable repression of homeotic (Hox) genes by forming multimeric protein complexes. We show (1) physical interaction between components of the U2 small nuclear ribonucleoprotein particle (U2 snRNP), including Sf3b1 and PcG proteins Zfp144 and Rnf2; and (2) that Sf3b1 heterozygous mice exhibit skeletal transformations concomitant with ectopic Hox expressions. These alterations are enhanced by Zfp144 mutation but repressed by Mll mutation (a trithorax-group gene). Importantly, the levels of Sf3b1 in PcG complexes were decreased in Sf3b1-heterozygous embryos. These findings suggest that Sf3b1-PcG protein interaction is essential for true PcG-mediated repression of Hox genes.
Assuntos
Osso e Ossos/embriologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/biossíntese , Fosfoproteínas/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Mutação , Proteína de Leucina Linfoide-Mieloide , Complexo Repressor Polycomb 1 , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proto-Oncogenes/genética , Fatores de Processamento de RNA , Proteínas Repressoras , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína LigasesRESUMO
Xenopus NO38 is an abundant nucleolar chaperone and a member of the nucleoplasmin (Np) family. Here, we report high-resolution crystal structures of the N-terminal domain of NO38, as a pentamer and a decamer. As expected, NO38 shares the Np family fold. In addition, NO38- and Np-core pentamers each use highly conserved residues and numerous waters to form their respective decamers. Further studies show that NO38 and Np each bind equal amounts of the four core histones. However, NO38 prefers the (H3-H4)(2) tetramer, while Np probably prefers H2A-H2B dimers. We also show that NO38 and Np will each bind noncognate histones when the preferred partner is absent. We suggest that these chaperones must form decamers in order to bind histones and differentiate between histone tetramers and dimers. When taken together, these data imply that NO38 may function as a histone chaperone in the nucleolus.
Assuntos
Nucléolo Celular/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Nucleofosmina , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , XenopusRESUMO
NO38 is a multidomain protein that belongs to the nucleoplasmin (Np) family. Previous studies have suggested that acidic chaperones such as Np may function as histone-storage platforms. Here, the purification and crystallization of the N-terminal domain of NO38 in two crystal forms is reported. The C2 crystal form diffracts to 1.9 A and contains two pentamers in the asymmetric unit, while the P1 crystals diffract to 1.7 A and contain a non-crystallographic decamer with 522 symmetry. By analogy with Np, the NO38 decamer may represent the active form of this chaperone.
Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Animais , Sítios de Ligação , Cálcio/química , Nucléolo Celular , Cristalografia por Raios X , DNA Complementar/metabolismo , Dimerização , Magnésio/química , Modelos Moleculares , Nucleofosmina , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Difração de Raios X , Xenopus laevisRESUMO
It has recently become clear that the nucleolus, the most prominent nuclear subcompartment, harbors diverse functions beyond its classic role in ribosome biogenesis. To gain insight into nucleolar functions, we have purified amplified nucleoli from Xenopus laevis oocytes using a novel approach involving fluorescence-activated cell sorting techniques. The resulting protein fraction was analyzed by mass spectrometry and used for the generation of monoclonal antibodies directed against nucleolar components. Here, we report the identification and molecular characterization of a novel, ubiquitous protein, which in most cell types appears to be a constitutive nucleolar component. Immunolocalization studies have revealed that this protein, termed NO66, is highly conserved during evolution and shows in most cells analyzed a dual localization pattern, i.e., a strong enrichment in the granular part of nucleoli and in distinct nucleoplasmic entities. Colocalizations with proteins Ki-67, HP1alpha, and PCNA, respectively, have further shown that the staining pattern of NO66 overlaps with certain clusters of late replicating chromatin. Biochemical experiments have revealed that protein NO66 cofractionates with large preribosomal particles but is absent from cytoplasmic ribosomes. We propose that in addition to its role in ribosome biogenesis protein NO66 has functions in the replication or remodeling of certain heterochromatic regions.