ABSTRACT
The transcriptional machinery is thought to dissociate from DNA during replication. Certain proteins, termed epigenetic marks, must be transferred from parent to daughter DNA strands in order to maintain the memory of transcriptional states1,2. These proteins are believed to re-initiate rebuilding of chromatin structure, which ultimately recruits RNA polymerase II (Pol II) to the newly replicated daughter strands. It is believed that Pol II is recruited back to active genes only after chromatin is rebuilt3,4. However, there is little experimental evidence addressing the central questions of when and how Pol II is recruited back to the daughter strands and resumes transcription. Here we show that immediately after passage of the replication fork, Pol II in complex with other general transcription proteins and immature RNA re-associates with active genes on both leading and lagging strands of nascent DNA, and rapidly resumes transcription. This suggests that the transcriptionally active Pol II complex is retained in close proximity to DNA, with a Pol II-PCNA interaction potentially underlying this retention. These findings indicate that the Pol II machinery may not require epigenetic marks to be recruited to the newly synthesized DNA during the transition from DNA replication to resumption of transcription.
Subject(s)
Chromatin , DNA Replication , DNA , Genes , RNA Polymerase II , Transcription, Genetic , Chromatin/genetics , DNA/biosynthesis , DNA/genetics , DNA/metabolism , DNA Polymerase II/metabolism , Epigenesis, Genetic , Proliferating Cell Nuclear Antigen/metabolism , RNA Polymerase II/metabolism , Transcription Factors, General/metabolism , RNA/genetics , RNA/metabolismABSTRACT
Propagation of gene-expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. We devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysines 4 or 27 is present during transcription but, surprisingly, is replaced by nonmethylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA Replication , Drosophila Proteins/metabolism , Drosophila/metabolism , Histone Code , Histones/metabolism , Animals , Drosophila/cytology , Drosophila/genetics , Embryo, Nonmammalian/metabolism , Epigenesis, Genetic , Polycomb Repressive Complex 1 , Proliferating Cell Nuclear Antigen/metabolism , Protein Processing, Post-Translational , S PhaseABSTRACT
Recruitment of transcription factors (TFs) to repressed genes in euchromatin is essential to activate new transcriptional programs during cell differentiation. However, recruitment of all TFs, including pioneer factors, is impeded by condensed H3K27me3-containing chromatin. Single-cell and gene-specific analyses revealed that, during the first hours of induction of differentiation of mammalian embryonic stem cells (ESCs), accumulation of the repressive histone mark H3K27me3 is delayed after DNA replication, indicative of a decondensed chromatin structure in all regions of the replicating genome. This delay provides a critical "window of opportunity" for recruitment of lineage-specific TFs to DNA. Increasing the levels of post-replicative H3K27me3 or preventing S phase entry inhibited recruitment of new TFs to DNA and significantly blocked cell differentiation. These findings suggest that recruitment of lineage-specifying TFs occurs soon after replication and is facilitated by a decondensed chromatin structure. This insight may explain the developmental plasticity of stem cells and facilitate their exploitation for therapeutic purposes.
Subject(s)
Cell Differentiation , Cell Lineage , Chromatin Assembly and Disassembly , Chromatin/metabolism , DNA Replication , DNA/biosynthesis , Embryonic Stem Cells/metabolism , Histones/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Animals , Binding Sites , Cell Plasticity , Chromatin/chemistry , DNA/chemistry , DNA/genetics , DNA Methylation , Gene Expression Regulation, Developmental , Histone Demethylases/metabolism , Histones/chemistry , Humans , Methylation , Mice , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Protein Binding , Structure-Activity Relationship , Time Factors , Transcription Factors/geneticsABSTRACT
The mechanism of epigenetic inheritance following DNA replication may involve dissociation of chromosomal proteins from parental DNA and reassembly on daughter strands in a specific order. Here we investigated the behaviour of different types of chromosomal proteins using newly developed methods that allow assessment of the assembly of proteins during DNA replication. Unexpectedly, most chromatin-modifying proteins tested, including methylases, demethylases, acetyltransferases and a deacetylase, are found in close proximity to PCNA or associate with short nascent DNA. Histone modifications occur in a temporal order following DNA replication, mediated by complex activities of different enzymes. In contrast, components of several major nucleosome-remodelling complexes are dissociated from parental DNA, and are later recruited to nascent DNA following replication. Epigenetic inheritance of gene expression patterns may require many aspects of chromatin structure to remain in close proximity to the replication complex followed by reassembly on nascent DNA shortly after replication.