Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Other
Summary
How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we develop the eSPAN method (enrichment and sequencing of protein associated nascent DNA) in mouse embryonic stem (ES) cells, which detects the distribution of histones with distinct modifications on leading and lagging strands in a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes primers used for synthesis of both leading and lagging strands, binds histone H3-H4 preferentially. The Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strand in both yeast and mouse ES cells. Finally, dysregulation of both coding genes and non-coding endogenous retroviruses (ERVs) is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication coupled processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.
Overall design
OK-Seq were performed in mouse ES (mES) cells to identify replication zones. The CUT&TAG and eSPAN experiments were conducted in WT, POLA1-2A mutant, MCM2-2A mutant, POLE3 KO and POLE4 KO mES cells. The histone markers performed by CUT&TAG and eSPAN methods in mES cells included H4K20me2, H4K12ac, H3K36me3. In Saccharomyces cerevisiae yeast, we also performed ChIP and eSPAN expriments for H3K4me3 and H3K56ac in WT and pol1-2A2 mutant yeast cells. For the regulation of gene expression, we performed total RNA-seq and H3K9me3 CUT&TAG experiments in WT, POLA1-2A mutant and MCM2-2A mutant mES cells.
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