RESUMO
We have recently discovered that the so-called subcortical maternal complex (SCMC) proteins composing of cytoplasmic lattices are destabilized in Uhrf1 knockout murine fully grown oocytes (FGOs). Here we report that human UHRF1 interacts with human NLRP5 and OOEP, which are core components of the SCMC. Moreover, NLRP5 and OOEP interact with DPPA3, which is an essential factor for exporting UHRF1 from the nucleus to the cytoplasm in oocytes. We identify that NLRP5, not OOEP, stabilizes UHRF1 protein in the cytoplasm utilizing specifically engineered cell lines mimicking UHRF1 status in oocytes and preimplantation embryos. Further, UHRF1 is destabilized both in the cytoplasm and nucleus of Nlrp5 knockout murine FGOs. Since pathogenic variants of the SCMC components frequently cause multilocus imprinting disturbance and UHRF1 is essential for maintaining CpG methylation of imprinting control regions during preimplantation development, our results suggest possible pathogenesis behind the disease, which has been a long-standing mystery.
RESUMO
Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is in most cases caused by mutations in either DNA methyltransferase (DNMT)3B, zinc finger and BTB domain containing 24, cell division cycle associated 7 or helicase lymphoid-specific. However, the causative genes of a few ICF patients remain unknown. We, herein, identified ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) as a novel causative gene of one such patient with atypical symptoms. This patient is a compound heterozygote for two previously unreported mutations in UHRF1: c.886C > T (p.R296W) and c.1852C > T (p.R618X). The R618X mutation plausibly caused nonsense-mediated decay, while the R296W mutation changed the higher order structure of UHRF1, which is indispensable for the maintenance of CG methylation along with DNMT1. Genome-wide methylation analysis revealed that the patient had a centromeric/pericentromeric hypomethylation, which is the main ICF signature, but also had a distinctive hypomethylation pattern compared to patients with the other ICF syndrome subtypes. Structural and biochemical analyses revealed that the R296W mutation disrupted the protein conformation and strengthened the binding affinity of UHRF1 with its partner LIG1 and reduced ubiquitylation activity of UHRF1 towards its ubiquitylation substrates, histone H3 and proliferating cell nuclear antigen -associated factor 15 (PAF15). We confirmed that the R296W mutation causes hypomethylation at pericentromeric repeats by generating the HEK293 cell lines that mimic the patient's UHRF1 molecular context. Since proper interactions of the UHRF1 with LIG1, PAF15 and histone H3 are essential for the maintenance of CG methylation, the mutation could disturb the maintenance process. Evidence for the importance of the UHRF1 conformation for CG methylation in humans is, herein, provided for the first time and deepens our understanding of its role in regulation of CG methylation.
Assuntos
Histonas , Doenças da Imunodeficiência Primária , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA/genética , DNA/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Células HEK293 , Histonas/genética , Histonas/metabolismo , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Mutação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Instabilidade Cromossômica/genética , Instabilidade Cromossômica/fisiologia , Centrômero/genética , Centrômero/metabolismo , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/metabolismo , Face/anormalidades , Genoma Humano/genética , Genoma Humano/fisiologiaRESUMO
Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Despite the numerous studies on the inhibition of amyloid formation, the prevention and treatment of a majority of amyloid-related disorders are still challenging. In this study, we investigated the effects of various plant extracts on amyloid formation of α-synuclein. We found that the extracts from Eucalyptus gunnii are able to inhibit amyloid formation, and to disaggregate preformed fibrils, in vitro. The extract itself did not lead cell damage. In the extract, miquelianin, which is a glycosylated form of quercetin and has been detected in the plasma and the brain, was identified and assessed to have a moderate inhibitory activity, compared to the effects of ellagic acid and quercetin, which are strong inhibitors for amyloid formation. The properties of miquelianin provide insights into the mechanisms controlling the assembly of α-synuclein in the brain.
RESUMO
Complex congenital chromosome abnormalities are rare but often cause severe symptoms. However, the structures and biological impacts of such abnormalities have seldomly been analyzed at the molecular level. Previously, we reported a Japanese female patient with severe developmental defects. The patient had an extra dicentric chromosome 21 (chr21) consisting of two partial chr21 copies fused together within their long arms along with two centromeres and many copy number changes. In this study, we performed whole-genome, transcriptional, and DNA methylation analyses, coupled with novel bioinformatic approaches, to reveal the complex structure of the extra chromosome and its transcriptional and epigenetic changes. Long-read sequencing accurately identified the structures of junctions related to the copy number changes in extra chr21 and suggested the mechanism of the structural changes. Our transcriptome analysis showed the overexpression of genes in extra chr21. Additionally, an allele-specific DNA methylation analysis of the long-read sequencing data suggested that the centromeric region of extra chr21 was hypermethylated, a property associated with the inactivation of one centromere in the extra chromosome. Our comprehensive analysis provides insights into the molecular mechanism underlying the generation of the extra chromosome and its pathogenic roles.
Assuntos
Centrômero , Epigênese Genética , Humanos , Feminino , Centrômero/genética , Cromossomos Humanos Par 21/genéticaRESUMO
Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is characterized by frequent appearance of multiradial chromosomes, which are distinctive chromosome fusions that occur at hypomethylated pericentromeric regions comprising repetitive sequences, in activated lymphocytes. The syndrome is caused by mutations in DNMT3B, ZBTB24, CDCA7, or HELLS. De novo DNA methylation is likely defective in patients with ICF syndrome harboring mutations in DNMT3B, whereas accumulating evidence suggests that replication-uncoupled maintenance DNA methylation of late-replicating regions is impaired in patients with ICF syndrome harboring mutations in ZBTB24, CDCA7, or HELLS. ZBTB24 is a transcriptional activator of CDCA7, and CDCA7 and HELLS compose a chromatin remodeling complex and are involved in the maintenance DNA methylation through an interaction with UHRF1 in a feed-forward manner. Furthermore, our recent studies possibly provided the missing link between DNA hypomethylation and the formation of the abnormal chromosomes; it could occur via aberrant transcription from the hypomethylated regions, followed by pathological R-loop formation. The homologous-recombination dominant condition caused by a defect in nonhomologous end joining observed in several types of ICF syndrome could facilitate the formation of multiradial chromosomes. Here, the latest knowledge regarding maintenance DNA methylation and chromosome stability provided by those studies is reviewed.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Instabilidade Cromossômica/genética , Metilação de DNA/genética , Replicação do DNA/genética , Face/anormalidades , Doenças da Imunodeficiência Primária/genética , Humanos , Modelos BiológicosRESUMO
The UHRF protein family consists of multidomain regulatory proteins that sense modification status of DNA and/or proteins and catalyze the ubiquitylation of target proteins. Through their functional domains, they interact with other molecules and serve as a hub for regulatory networks of several important biological processes, including maintenance of DNA methylation and DNA damage repair. The UHRF family is conserved in vertebrates and plants but is missing from fungi and many nonvertebrate animals. Mammals commonly have UHRF1 and UHRF2, but, despite their high structural similarity, the two paralogues appear to have distinct functions. Furthermore, UHRF1 and UHRF2 show different expression patterns and different outcomes in gene knockout experiments. In this review, we summarize the current knowledge on the molecular function of the UHRF family in various biological pathways and discuss their roles in epigenetics, development, gametogenesis, and carcinogenesis, with a focus on the mammalian UHRF proteins.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Ubiquitina-Proteína Ligases , Animais , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Carcinogênese/genética , DNA , Metilação de DNA , Epigênese Genética , Mamíferos/genética , Mamíferos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: No effective molecular targeted therapy has been established for SCC. We conducted a comprehensive study of SCC patients using RNA-sequencing and TCGA dataset to clarify the driver oncogene of SCC. METHOD: Forty-six samples of 23 patients were totally analyzed with RNA-sequencing. We then searched for candidate-oncogenes of SCC using the TCGA database. To identify candidate oncogenes, we used the following 2 criteria: (1) the genes of interest were overexpressed in tumor tissues of SCC patients in comparison to normal tissues; and (2) using an integrated mRNA expression and DNA copy number profiling analysis using the TCGA dataset, the DNA copy number of the genes was positively correlated with the mRNA expression. RESULT: We identified 188 candidate-oncogenes. Among those, the high expression of SLC38A7 was a strong prognostic marker that was significantly associated with a poor prognosis in terms of both overall survival (OS) and recurrence-free survival in the TCGA dataset (P < 0.05). Additionally, 202 resected SCC specimens were also subjected to an immunohistochemical analysis. Patients with the high expression of SLC38A7 (alternative name is sodium-coupled amino acid transporters 7) protein showed significantly shorter OS in comparison to those with the low expression of SLC38A7 protein [median OS 3.9âyears (95% confidence interval, 2.4-6.4 years) vs 2.2âyears (95% confidence interval, 1.9-4.1 years); log rank test: P = 0.0021]. CONCLUSION: SLC38A7, which is the primary lysosomal glutamine transporter required for the extracellular protein-dependent growth of cancer cells, was identified as a candidate therapeutic target of SCC.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Terapia de Alvo Molecular , Idoso , Sistema A de Transporte de Aminoácidos , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/cirurgia , Variações do Número de Cópias de DNA , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Oncogenes/genética , Prognóstico , RNA Mensageiro/metabolismo , Estudos RetrospectivosRESUMO
Monoallelic gene expression occurs in various mammalian cells and can be regulated genetically, epigenetically and/or stochastically. We identified 145 monoallelically expressed genes (MoEGs), including seven known imprinted genes, in mouse embryonic stem cells (ESCs) derived from reciprocal F1 hybrid blastocysts and cultured in 2i/LIF. As all MoEGs except for the imprinted genes were expressed in a genetic-origin-dependent manner, we focused on this class of MoEGs for mechanistic studies. We showed that a majority of the genetic-origin-dependent MoEGs identified in 2i/LIF ESCs remain monoallelically expressed in serum/LIF ESCs, but become more relaxed or even biallelically expressed upon differentiation. These MoEGs and their regulatory regions were highly enriched for single nucleotide polymorphisms. In addition, some MoEGs were associated with retrotransposon insertions/deletions, consistent with the fact that certain retrotransposons act as regulatory elements in pluripotent stem cells. Interestingly, most MoEGs showed allelic differences in enrichment of histone H3K27me and H3K4me marks, linking allelic epigenetic differences and monoallelic expression. In contrast, there was little or no allelic difference in CpG methylation or H3K9me. Taken together, our study highlights the impact of genetic variation including single nucleotide polymorphisms and retrotransposon insertions/deletions on monoallelic epigenetic marks and expression in ESCs.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Embrionárias Murinas/metabolismo , Transcriptoma/genética , Alelos , Animais , Diferenciação Celular/genética , Linhagem Celular , Metilação de DNA/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética/genética , Epigenômica/métodos , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Impressão Genômica/genética , Masculino , Camundongos , Camundongos Endogâmicos , Células-Tronco Pluripotentes/metabolismoRESUMO
DNA methylation (DNAme) profiling is used to establish specific biomarkers to improve the diagnosis of patients with inherited neurodevelopmental disorders and to guide mutation screening. In the specific case of mendelian disorders of the epigenetic machinery, it also provides the basis to infer mechanistic aspects with regard to DNAme determinants and interplay between histone and DNAme that apply to humans. Here, we present comparative methylomes from patients with mutations in the de novo DNA methyltransferases DNMT3A and DNMT3B, in their catalytic domain or their N-terminal parts involved in reading histone methylation, or in histone H3 lysine (K) methylases NSD1 or SETD2 (H3 K36) or KMT2D/MLL2 (H3 K4). We provide disease-specific DNAme signatures and document the distinct consequences of mutations in enzymes with very similar or intertwined functions, including at repeated sequences and imprinted loci. We found that KMT2D and SETD2 germline mutations have little impact on DNAme profiles. In contrast, the overlapping DNAme alterations downstream of NSD1 or DNMT3 mutations underlines functional links, more specifically between NSD1 and DNMT3B at heterochromatin regions or DNMT3A at regulatory elements. Together, these data indicate certain discrepancy with the mechanisms described in animal models or the existence of redundant or complementary functions unforeseen in humans.
Assuntos
Metilação de DNA/genética , Doenças Genéticas Inatas/genética , Histonas/genética , Mutação , Doenças Raras/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Doenças Genéticas Inatas/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/genética , Doenças Raras/metabolismo , DNA Metiltransferase 3BRESUMO
Immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disorder caused by mutations in either DNMT3B, ZBTB24, CDCA7, HELLS or an unknown gene(s). Among the known causative genes, ZBTB24 encodes a member of the BTB-zinc finger (ZF) transcription factor family. The protein possesses a BTB domain, an AT-hook and eight C2H2 ZF motifs. All ZBTB24 mutations reported in ICF patients are predicted to disrupt at least one ZF motif. Here, we show that both AT-hook and distinct ZF motifs, particularly the 6th motif, of human and mouse ZBTB24 proteins are important for their heterochromatin localization. On the other hand, the 6th and 7th ZF motifs, and not the AT-hook or the BTB domain, of the human and mouse proteins are essential for transcriptional activation of CDCA7, another ICF causative gene and a known target of ZBTB24. By deletion analysis of the human CDCA7 promoter, we show that two motifs for ZBTB24 binding are important for transcriptional activation of this gene. These results reveal the evolutionarily conserved domains and motifs important for the biological function of ZBTB24, which provides a basis for understanding the molecular mechanisms underlying the pathogenesis of ICF syndrome.
Assuntos
Motivos de Aminoácidos , Heterocromatina , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/isolamento & purificação , Ativação Transcricional , Animais , Dedos de Zinco CYS2-HIS2 , Face/anormalidades , Células HEK293 , Humanos , Camundongos , Mutação , Células NIH 3T3 , Proteínas Nucleares , Doenças da Imunodeficiência Primária/genética , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Dedos de ZincoRESUMO
The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos.
Assuntos
Blastocisto/metabolismo , Metilação de DNA , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Reprogramação Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Desenvolvimento Embrionário , Epigênese Genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Oócitos/crescimento & desenvolvimento , Oogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Multiple covalent modifications on a histone tail are often recognized by linked histone reader modules. UHRF1 [ubiquitin-like, containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1], an essential factor for maintenance of DNA methylation, contains linked two-histone reader modules, a tandem Tudor domain and a PHD finger, tethered by a 17-aa linker, and has been implicated to link histone modifications and DNA methylation. Here, we present the crystal structure of the linked histone reader modules of UHRF1 in complex with the amino-terminal tail of histone H3. Our structural and biochemical data provide the basis for combinatorial readout of unmodified Arg-2 (H3-R2) and methylated Lys-9 (H3-K9) by the tandem tudor domain and the PHD finger. The structure reveals that the intermodule linker plays an essential role in the formation of a histone H3-binding hole between the reader modules by making extended contacts with the tandem tudor domain. The histone H3 tail fits into the hole by adopting a compact fold harboring a central helix, which allows both of the reader modules to simultaneously recognize the modification states at H3-R2 and H3-K9. Our data also suggest that phosphorylation of a linker residue can modulate the relative position of the reader modules, thereby altering the histone H3-binding mode. This finding implies that the linker region plays a role as a functional switch of UHRF1 involved in multiple regulatory pathways such as maintenance of DNA methylation and transcriptional repression.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Histonas/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Subunidades Proteicas/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Cromatografia Líquida , Clonagem Molecular , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli , Humanos , Espectroscopia de Ressonância Magnética , Fosforilação , Reação em Cadeia da Polimerase , Ligação Proteica , Espectrometria de Massas em Tandem , Ubiquitina-Proteína LigasesRESUMO
JMJD6 is reported to hydroxylate lysyl residues of a splicing factor, U2AF65. In this study, we found that JMJD6 hydroxylates histone lysyl residues. In vitro experiments showed that JMJD6 has a binding affinity to histone proteins and hydroxylates multiple lysyl residues of histone H3 and H4 tails. Using JMJD6 knock-out mouse embryos, we revealed that JMJD6 hydroxylates lysyl residues of histones H2A/H2B and H3/H4 in vivo by amino acid composition analysis. 5-Hydroxylysine was detected at the highest level in histones purified from murine testis, which expressed JMJD6 at a significantly high level among various tissues examined, and JMJD6 overexpression increased the amount of 5-hydroxylysine in histones in human embryonic kidney 293 cells. These results indicate that histones are additional substrates of JMJD6 in vivo. Because 5-hydroxylation of lysyl residues inhibited N-acetylation and N-methylation by an acetyltransferase and a methyltransferase, respectively, in vitro, histone 5-hydroxylation may have important roles in epigenetic regulation of gene transcription or chromosomal rearrangement.
Assuntos
Cromossomos de Mamíferos/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Transcrição Gênica/fisiologia , Acetilação , Animais , Cromossomos de Mamíferos/genética , Células HEK293 , Histonas/genética , Humanos , Hidroxilação/fisiologia , Histona Desmetilases com o Domínio Jumonji/genética , Lisina/genética , Masculino , Metilação , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Testículo/enzimologiaRESUMO
Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a rare autosomal recessive disorder, manifests with hypoglobulinemia and chromosomal instability accompanied by DNA hypomethylation. Pathological variants in the DNMT3B, ZBTB24, CDCA7, or HELLS genes underlie its etiology. Activated lymphocytes from patients often display distinctive multiradial chromosomes fused via pericentromeric regions. Recent studies have provided deeper insights into how pathological variants in ICF-related proteins cause DNA hypomethylation and chromosome instability. However, the understanding of the molecular pathogenesis underlying immunodeficiency is still in its nascent stages. In the past half-decade, the roles of CDCA7, HELLS, and ZBTB24 in classical non-homologous end joining during double-strand DNA break repair and immunoglobulin class-switch recombination (CSR) have been unveiled. Nevertheless, given the decreased all classes of immunoglobulins in most patients, CSR deficiency alone cannot fully account for the immunodeficiency. The latest finding showing dysregulation of immunoglobulin signaling may provide a clue to understanding the immunodeficiency mechanism. While less common, a subgroup of patients exhibits T-cell abnormalities alongside B-cell anomalies, including reduced regulatory T-cells and increased effector memory T- and follicular helper T-cells. The dysregulation of immunoglobulin signaling in B-cells, the imbalance in T-cell subsets, and/or satellite RNA-mediated activation of innate immune response potentially explain autoimmune manifestations in a subset of patients. These findings emphasize the pivotal roles of ICF-related proteins in both B- and T-cell functions. ICF syndrome studies have illuminated many fundamental mechanisms. Further investigations will certainly continue to unveil additional mechanisms and their interplay.
Assuntos
Reparo do DNA , Epigênese Genética , Síndromes de Imunodeficiência , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Metilação de DNA , Animais , Switching de Imunoglobulina/genética , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/imunologia , Doenças da Imunodeficiência Primária/diagnósticoRESUMO
[This corrects the article DOI: 10.3389/fimmu.2024.1405022.].
RESUMO
Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is a rare autosomal recessive disorder that shows DNA hypomethylation at pericentromeric satellite-2 and -3 repeats in chromosomes 1, 9 and 16. ICF syndrome is classified into two groups: type 1 (ICF1) patients have mutations in the DNMT3B gene and about half of type 2 (ICF2) patients have mutations in the ZBTB24 gene. Besides satellite-2 and -3 repeats, α-satellite repeats are also hypomethylated in ICF2. In this study, we report three novel ZBTB24 mutations in ICF2. A Japanese patient was homozygous for a missense mutation (C383Y), and a Cape Verdean patient was compound heterozygous for a nonsense mutation (K263X) and a frame-shift mutation (C327W fsX54). In addition, the second Japanese patient was homozygous for a previously reported nonsense mutation (R320X). The C383Y mutation abolished a C2H2 motif in one of the eight zinc-finger domains, and the other three mutations caused a complete or large loss of the zinc-finger domains. Our immunofluorescence analysis revealed that mouse Zbtb24 proteins possessing a mutation corresponding to either C383Y or R320X are mislocalized from pericentrometic heterochromatin, suggesting the importance of the zinc-finger domains in proper intranuclear localization of this protein. We further revealed that the proper localization of wild-type Zbtb24 protein does not require DNA methylation.
Assuntos
Povo Asiático/genética , População Negra/genética , Face/anormalidades , Síndromes de Imunodeficiência/genética , Proteínas Repressoras/genética , Adolescente , Adulto , Animais , Linhagem Celular , Centrômero/metabolismo , Pré-Escolar , Aberrações Cromossômicas , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 1/metabolismo , Cromossomos Humanos Par 16/genética , Clonagem Molecular , Metilação de DNA , Feminino , Genômica , Humanos , Síndromes de Imunodeficiência/diagnóstico , Masculino , Camundongos , Mutação , Células NIH 3T3 , Doenças da Imunodeficiência Primária , Proteínas Recombinantes de Fusão/genética , Análise de Sequência , Dedos de Zinco/genéticaRESUMO
Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a protein essential for the maintenance of DNA methylation in somatic cells. However, UHRF1 is predominantly localized in the cytoplasm of mouse oocytes and preimplantation embryos, where it may play a role unrelated to the nuclear function. We herein report that oocyte-specific Uhrf1 KO results in impaired chromosome segregation, abnormal cleavage division, and preimplantation lethality of derived embryos. Our nuclear transfer experiment showed that the phenotype is attributable to cytoplasmic rather than nuclear defects of the zygotes. A proteomic analysis of KO oocytes revealed the down-regulation of proteins associated with microtubules including tubulins, which occurred independently of transcriptomic changes. Intriguingly, cytoplasmic lattices were disorganized, and mitochondria, endoplasmic reticulum, and components of the subcortical maternal complex were mislocalized. Thus, maternal UHRF1 regulates the proper cytoplasmic architecture and function of oocytes and preimplantation embryos, likely through a mechanism unrelated to DNA methylation.
Assuntos
Oócitos , Proteômica , Animais , Camundongos , Citosol , Retículo Endoplasmático , Mitocôndrias , Proteínas Estimuladoras de Ligação a CCAAT/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
A number of histone demethylases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human disease like cancer have not been well understood. Here, we demonstrate important roles of lysine-specific demethylase 1 (LSD1) in human carcinogenesis. Expression levels of LSD1 are significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001). cDNA microarray analysis also revealed its transactivation in lung and colorectal carcinomas. LSD1-specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of various bladder and lung cancer cell lines. Concordantly, introduction of exogenous LSD1 expression promoted cell cycle progression of human embryonic kidney fibroblast cells. Expression profile analysis showed that LSD1 could affect the expression of genes involved in various chromatin-modifying pathways such as chromatin remodeling at centromere, centromeric heterochromatin formation and chromatin assembly, indicating its essential roles in carcinogenesis through chromatin modification.
Assuntos
Cromatina/fisiologia , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/genética , Neoplasias/genética , Neoplasias da Bexiga Urinária/genética , Ciclo Celular , Divisão Celular , Linhagem Celular Tumoral , Cromatina/genética , DNA Complementar/genética , DNA de Neoplasias/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Bexiga Urinária/citologia , Bexiga Urinária/fisiologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Protein arginine methylation is a novel post-translational modification regulating a diversity of cellular processes, including histone functions, but the roles of protein arginine methyltransferases (PRMTs) in human cancer are not well investigated. To address this issue, we first examined expression levels of genes belonging to the PRMT family and found significantly higher expression of PRMT1 and PRMT6, both of which are Type I PRMTs, in cancer cells of various tissues than in non-neoplastic cells. Abrogation of the expression of these genes with specific siRNAs significantly suppressed growth of bladder and lung cancer cells. Expression profile analysis using the cells transfected with the siRNAs indicated that PRMT1 and PRMT6 interplay in multiple pathways, supporting regulatory roles in the cell cycle, RNA processing and also DNA replication that are fundamentally important for cancer cell proliferation. Furthermore, we demonstrated that serum asymmetric dimethylarginine (ADMA) levels of a number of cancer cases are significantly higher than those of nontumor control cases. In summary, our results suggest that dysregulation of PRMT1 and PRMT6 can be involved in human carcinogenesis and that these Type I arginine methyltransferases are good therapeutic targets for various types of cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Neoplasias da Bexiga Urinária/genética , Antracenos , Arginina/análogos & derivados , Arginina/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Divisão Celular , Linhagem Celular Tumoral , Replicação do DNA , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Propano/análogos & derivados , Propano/sangue , RNA Interferente Pequeno/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
We have previously reported a novel homozygous 4-bp deletion in DDHD1 as the responsible variant for spastic paraplegia type 28 (SPG28; OMIM#609340). The variant causes a frameshift, resulting in a functionally null allele in the patient. DDHD1 encodes phospholipase A1 (PLA1) catalyzing phosphatidylinositol to lysophosphatidylinositol (LPI). To clarify the pathogenic mechanism of SPG28, we established Ddhd1 knockout mice (Ddhd1[-/-]) carrying a 5-bp deletion in Ddhd1, resulting in a premature termination of translation at a position similar to that of the patient. We observed a significant decrease in foot-base angle (FBA) in aged Ddhd1(-/-) (24 months of age) and a significant decrease in LPI 20:4 (sn-2) in Ddhd1(-/-) cerebra (26 months of age). These changes in FBA were not observed in 14 months of age. We also observed significant changes of expression levels of 22 genes in the Ddhd1(-/-) cerebra (26 months of age). Gene Ontology (GO) terms relating to the nervous system and cell-cell communications were significantly enriched. We conclude that the reduced signaling of LPI 20:4 (sn-2) by PLA1 dysfunction is responsible for the locomotive abnormality in SPG28, further suggesting that the reduction of downstream signaling such as GPR55 which is agonized by LPI is involved in the pathogenesis of SPG28.