ABSTRACT
RNA-binding proteins play a crucial role in the post-transcriptional regulation of gene expression. Polypyrimidine tract binding protein (PTB in humans) has been extensively characterized as an important splicing factor, and has additional functions in 3' end processing and translation. ROD1 is a PTB paralog containing four RRM (RNA recognition motif) domains. Here, we discover a function of ROD1 in nonsense-mediated mRNA decay (NMD). We show that ROD1 and the core NMD factor UPF1 interact and co-regulate an extensive number of target genes. Using a reporter system, we demonstrate that ROD1, similarly to UPF1 and UPF2, is required for the destabilization of a known NMD substrate. Finally, we show through RIP-seq that ROD1 and UPF1 associate with a significant number of common transcripts.
Subject(s)
Nonsense Mediated mRNA Decay , Polypyrimidine Tract-Binding Protein/metabolism , RNA, Messenger/metabolism , Codon, Nonsense , HEK293 Cells , Humans , Polypyrimidine Tract-Binding Protein/genetics , RNA StabilityABSTRACT
The Polycomb group (Pc-G) genes encode proteins that assemble into complexes implicated in the epigenetic maintenance of heritable patterns of expression of developmental genes, a function largely conserved from Drosophila to mammals and plants. The Pc-G is thought to act at the chromatin level to silence expression of target genes; however, little is known about the molecular basis of this repression. In keeping with the evidence that Pc-G homologs in higher vertebrates exist in related pairs, we report here the isolation of XPc1, a second Polycomb homolog in Xenopus laevis. We show that XPc1 message is maternally deposited in a translationally masked form in Xenopus oocytes, with XPc1 protein first appearing in embryonic nuclei shortly after the blastula stage. XPc1 acts as a transcriptional repressor in vivo when tethered to a promoter in Xenopus embryos. We find that XPc1-mediated repression can be only partially alleviated by an increase in transcription factor dosage and that inhibition of deacetylase activity by trichostatin A treatment has no effect on XPc1 repression, suggesting that histone deacetylation does not form the basis for Pc-G-mediated repression in our assay.
Subject(s)
Gene Expression Regulation, Developmental , Histone Deacetylases/physiology , Repressor Proteins/genetics , Transcription Factors , Transcription, Genetic , Xenopus Proteins , Xenopus laevis/embryology , Amino Acid Sequence , Animals , Blotting, Northern , Blotting, Western , Cell Nucleus/metabolism , Centrifugation, Density Gradient , Enzyme Inhibitors/pharmacology , Genes, Reporter , Hydroxamic Acids/pharmacology , Molecular Sequence Data , Phosphorylation , Sequence Homology, Amino Acid , Time Factors , Tissue DistributionABSTRACT
CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.
Subject(s)
Chromosomal Proteins, Non-Histone , DNA Methylation , DNA-Binding Proteins/metabolism , Histone Deacetylases/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins , Transcription, Genetic , Amino Acid Sequence , Animals , Binding Sites , Methyl-CpG-Binding Protein 2 , Molecular Sequence Data , Transcription Factors/metabolism , Xenopus Proteins , Xenopus laevisABSTRACT
We have used gene competition to distinguish between possible mechanisms of transcriptional activation of the genes of the human beta-globin locus. The insertion of a second beta-globin gene at different points in the locus shows that the more proximal beta gene competes more effectively for activation by the locus control region (LCR). Reducing the relative distance between the genes and the LCR reduces the competitive advantage of the proximal gene, a result that supports activation by direct interaction between the LCR and the genes. Visualization of the primary transcripts shows that the level of transcription is proportional to the frequency of transcriptional periods and that such periods last approximately 8 min in vivo. We also find that the position of the beta-globin gene in the locus is important for correct developmental regulation.
Subject(s)
Chromatin/metabolism , Globins/genetics , Transcriptional Activation/physiology , Animals , Binding, Competitive/genetics , Cosmids , Gene Expression Regulation, Developmental , Humans , Kinetics , Mice , Mice, Transgenic , Mutagenesis/physiology , Oocytes , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transgenes/geneticsABSTRACT
Locus control regions (LCRs) are responsible for initiating and maintaining a stable tissue-specific open chromatin structure of a locus. In transgenic mice, LCRs confer high level expression on linked genes independent of position in the mouse genome. Here we show that an incomplete LCR loses this property when integrated into heterochromatic regions. Two disruption mechanisms were observed. One is classical position-effect variegation, resulting in continuous transcription in a clonal subpopulation of cells. The other is a novel mechanism resulting in intermittent gene transcription in all cells. We conclude that only a complete LCR fully overcomes heterochromatin silencing and that it controls the level of transcription by ensuring activity in all cells at all times rather than directly controlling the rate of transcription.
Subject(s)
Gene Expression Regulation/genetics , Heterochromatin/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription, Genetic/genetics , Animals , Centromere/genetics , Chromosome Mapping , Deoxyribonuclease I , Deoxyribonucleases, Type II Site-Specific , Erythrocytes/chemistry , Gene Dosage , Globins/genetics , Humans , Mice , Mice, Transgenic , RNA, Messenger/analysis , Sequence Deletion , Single-Strand Specific DNA and RNA Endonucleases , Time Factors , Transgenes/geneticsABSTRACT
Recent applications of cell biology and molecular genetics have built an image of nuclear organization in which the molecular machines involved in transcription, RNA processing and replication assemble morphologically distinct nuclear organelles with defined functional properties. These observations indicate a very high level of structural organization for the various metabolic activities occurring within the nucleus. We discuss the possible existence of novel regulatory functions inherent to nuclear architecture itself.
Subject(s)
Cell Compartmentation/physiology , Cell Nucleus/physiology , Animals , Cell Nucleolus/physiology , DNA Replication , Leukemia, Myeloid, Acute/pathology , Models, Biological , RNA, Messenger/biosynthesisABSTRACT
The precise time of appearance of the first hematopoietic stem cell activity in the developing mouse embryo is unknown. Recently the aorta-gonad-mesonephros region of the developing mouse embryo has been shown to possess hematopoietic colony-forming activity (CFU-S) in irradiated recipient mice. To determine whether the mouse embryo possesses definitive hematopoietic stem cell activity in the analogous AGM region and to determine the order of appearance of stem cells in the yolk sac, AGM region, and liver, we transferred these embryonic tissues into adult irradiated recipients. We report here the long-term, complete, and functional hematopoietic repopulation of primary and serial recipients with AGM-derived cells. We observe potent hematopoietic stem cell activity in the AGM region before the appearance of yolk sac and liver stem cell activity and discuss a model for the maturation of stem cell activity in mouse embryogenesis.
Subject(s)
Embryo, Mammalian/cytology , Hematopoietic Stem Cells/cytology , Nuclear Proteins , Transcription Factors , Animals , Aorta/cytology , Aorta/embryology , Base Sequence , Colony-Forming Units Assay , DNA Primers/genetics , DNA-Binding Proteins/genetics , Embryonic and Fetal Development/genetics , Female , Fetal Tissue Transplantation , Gestational Age , Gonads/cytology , Gonads/embryology , Hematopoiesis/genetics , Male , Mesonephros/cytology , Mesonephros/embryology , Mice , Molecular Sequence Data , Myogenin/genetics , Pregnancy , Sex-Determining Region Y ProteinABSTRACT
We have used a linker-based ligation strategy to combine two 35-kb cosmid inserts from the human beta-globin locus into one linear fragment containing the entire locus. This 70-kb fragment was introduced into transgenic mice by microinjection of fertilized eggs. Southern blot analysis showed that a single complete transgene locus can be introduced into the germ line with high efficiency. Analysis of the expression patterns of the locus during development shows that the epsilon-globin gene behaves as a purely embryonic gene, the gamma-globin gene as an embryonic and early fetal gene, and the beta-globin gene as a fetal adult gene. Quantitation of expression showed that the levels of transcription of the epsilon- and gamma-globin genes are reversed relative to their mouse homologs but that the total output of the human and mouse loci is constant during development. These results suggest that multiple changes in DNA sequences and transcription factor balance must have occurred for the human gamma-globin gene to have evolved into a fetal gene.
