RESUMO
To realize highly sensitive immunoassays, high optical density probes conjugated with antibodies for target antigens have been demanded in order to increase the visibility of antigen-antibody complex formation. We herein demonstrate the development of an immunoassay system using magnetic and fluorescent Janus particles as probes in conjunction with an antibody-immobilized microfluidic device. The concentration of the detection limit at which there was a significant difference between SARS-CoV-2 and human coronavirus 229E antigens was 3.1 ng/mL, and the standard deviation of the signal was less than 5%. The immunofluorescent probe and immunoassay system developed in this study are expected to be applicable not only to SARS-CoV-2 but also to the quantitative measurement of various other disease marker proteins and biomolecules.
RESUMO
Transcription elongation is stimulated by positive transcription elongation factor b (P-TEFb), for which activity is repressed in the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. We show here a critical role of 7SK snRNP in growth control of primordial germ cells (PGCs). The expression of p15(INK4b), a cyclin-dependent kinase inhibitor (CDKI) gene, in PGCs is selectively activated by P-TEFb and its recruiting molecule, Brd4, when the amount of active P-TEFb is increased due to reduction of the 7SK snRNP, and PGCs consequently undergo growth arrest. These results indicate that CDKI gene-specific control of transcription by 7SK snRNP plays a pivotal role in the maintenance of PGC proliferation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes cdc/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Proteínas Nucleares/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In embryonic stem cells (ESCs), the expression of development-related genes, including germ cell-related genes, is globally repressed. The transcription factor MAX represses germ cell-related gene expression in ESCs via PCGF6-polycomb repressive complex 1 (PRC1), which consists of several epigenetic factors. However, we predicted that MAX represses germ cell-related gene expression through several additional mechanisms because PCGF6-PRC1 regulates the expression of only a subset of genes repressed by MAX. Here, we report that MAX associated with DNA methyltransferases (DNMTs) and the histone methyltransferase SETDB1 cooperatively control germ cell-related gene expression in ESCs. Both DNA methylation and histone H3 lysine 9 tri-methylation of the promoter regions of several germ cell-related genes were not affected by knockout of the PRC1 components, indicating that the MAX-DNMT and MAX-SETDB1 pathways are independent of the PCGF6-PRC1 pathway. Our findings provide insights into our understanding of MAX-based repressive mechanisms of germ cell-related genes in ESCs.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas Correpressoras/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Biomarcadores/metabolismo , Fracionamento Químico , Metilação de DNA/genética , Histonas/metabolismo , Lisina/metabolismo , Metilação , Camundongos , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Ligação ProteicaRESUMO
Embryonic stem cells and primordial germ cells (PGCs) express many pluripotency-associated genes, but embryonic stem cells do not normally undergo conversion into primordial germ cells. Thus, we predicted that there is a mechanism that represses primordial germ cell-related gene expression in embryonic stem cells. Here we identify genes involved in this putative mechanism, by using an embryonic stem cell line with a Vasa reporter in an RNA interference screen of transcription factor genes expressed in embryonic stem cells. We identify five genes that result in the expression of Vasa when silenced. Of these, Max is the most striking. Transcriptome analysis reveals that Max knockdown in embryonic stem cells results in selective, global derepression of germ cell-specific genes. Max interacts with histone H3K9 methyltransferases and associates with the germ cell-specific genes in embryonic stem cells. In addition, Max knockdown results in a decrease in histone H3K9 dimethylation at their promoter regions. We propose that Max is part of protein complex that acts as a repressor of germ cell-related genes in embryonic stem cells.