Abstract
REV1-deficient chicken DT40 cells are compromised in replicating G quadruplex (G4)-forming DNA. This results in localised, stochastic loss of parental chromatin marks and changes in gene expression. We previously proposed that this epigenetic instability arises from G4-induced replication fork stalls disrupting the accurate propagation of chromatin structure through replication. Here, we test this model by showing that a single G4 motif is responsible for the epigenetic instability of the BU-1 locus in REV1-deficient cells, despite its location 3.5 kb from the transcription start site (TSS). The effect of the G4 is dependent on it residing on the leading strand template, but is independent of its in vitro thermal stability. Moving the motif to more than 4 kb from the TSS stabilises expression of the gene. However, loss of histone modifications (H3K4me3 and H3K9/14ac) around the transcription start site correlates with the position of the G4 motif, expression being lost only when the promoter is affected. This supports the idea that processive replication is required to maintain the histone modification pattern and full transcription of this model locus.
Highlights
The vertebrate genome is littered with sequences capable of forming secondary structures that can interfere with replication (Mirkin & Mirkin, 2007), of which the G quadruplex (G4) is one of the most intensively studied (Maizels & Gray, 2013). dG bases in G-rich sequences can form planar Hoogsteen base-paired rings (G quartets) that stack to form often highly thermodynamically stable secondary structures, G quadruplexes (Sundquist & Klug, 1989; Williamson et al, 1989)
We have previously shown that expression of Bu-1a is unstable in DT40 mutants lacking enzymes involved in replicating G4s, including the specialised DNA polymerase REV1 and DNA helicases FANCJ, WRN and BLM (Sarkies et al, 2012)
We previously speculated that instability of Bu-1a expression in REV1-deficient cells would be dependent on the +3.5 G4 motif (Sarkies et al, 2012), since this structure would form on the leading strand template of a replication fork heading towards the transcription start site (TSS) from the 30 end of the locus (Fig 1B)
Summary
The vertebrate genome is littered with sequences capable of forming secondary structures that can interfere with replication (Mirkin & Mirkin, 2007), of which the G quadruplex (G4) is one of the most intensively studied (Maizels & Gray, 2013). dG bases in G-rich sequences can form planar Hoogsteen base-paired rings (G quartets) that stack to form often highly thermodynamically stable secondary structures, G quadruplexes (Sundquist & Klug, 1989; Williamson et al, 1989). A broader range of enzymes is involved These include the homologue of PIF1 (Sanders, 2010), other specialised helicases including FANCJ (Cheung et al, 2002; Kruisselbrink et al, 2008; London et al, 2008; Wu et al, 2008), WRN and BLM (Sun et al, 1998; Fry & Loeb, 1999; Mohaghegh et al, 2001) and the specialised DNA polymerases REV1 (Sarkies et al, 2010), Pol j and Pol g (Betous et al, 2009). We suggested, delayed chromatinisation of the gap would result in loss of the epigenetic marks carried by the histones resulting in changes in gene expression Consistent with this model, we have observed loss of histone marks associated with transcriptionally repressed chromatin (e.g. H3K9me2) and with transcriptionally active chromatin (e.g. H3K4me and H3K19/14ac) in loci containing G4 motifs (Sarkies et al, 2010, 2012). Our data provide direct evidence of a link between loss of histone modifications due to interruption of DNA replication and inability to maintain high-level gene expression
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