ABSTRACT
A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.
Subject(s)
Deoxyribonucleotides/analysis , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, DNA/methods , DNA-Directed DNA Polymerase/metabolism , Deoxyribonucleosides/chemistry , Deoxyribonucleotides/chemistry , Limit of Detection , Microscopy, Fluorescence , Oligodeoxyribonucleotides/biosynthesis , Oligodeoxyribonucleotides/chemistry , Sensitivity and SpecificityABSTRACT
Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos.
Subject(s)
Cellular Reprogramming , DNA Methylation , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Developmental , 5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/metabolism , Animals , CCAAT-Enhancer-Binding Proteins , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dioxygenases , Histones/metabolism , Mice , Models, Genetic , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Time Factors , Transfection , Ubiquitin-Protein LigasesABSTRACT
Endocytosis is essential for uptake of many substances into the cell, but how it links to nutritional signalling is poorly understood. Here, we show a new role for endocytosis in regulating the response to low phosphate in Schizosaccharomyces pombe. Loss of function of myosin I (Myo1), Sla2/End4 or Arp2, proteins involved in the early steps of endocytosis, led to increased proliferation in low-phosphate medium compared to controls. We show that once cells are deprived of phosphate they undergo a quiescence response that is dependent on the endocytic function of Myo1. Transcriptomic analysis revealed a wide perturbation of gene expression with induction of stress-regulated genes upon phosphate starvation in wild-type but not Δmyo1 cells. Thus, endocytosis plays a pivotal role in mediating the cellular response to nutrients, bridging the external environment and internal molecular functions of the cell.
Subject(s)
Endocytosis/physiology , Gene Expression Regulation, Fungal/physiology , Myosin Heavy Chains/metabolism , Phosphates/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Signal Transduction/physiology , Actin-Related Protein 2/genetics , Actin-Related Protein 2/metabolism , Gene Deletion , Myosin Heavy Chains/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , TranscriptomeABSTRACT
Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.
