Analysis of Human Syndromes with Disordered Chromatin Reveals the Impact of Heterochromatin on the Efficacy of ATM-Dependent G₂/M Checkpoint Arrest

Abstract

Heterochromatin (HC) poses a barrier to γH2AX focus expansion and DNA double-strand break (DSB) repair, the latter being relieved by ATM-dependent KAP-1 phosphorylation. Using high-resolution imaging, we show here that the HC superstructure markedly restricts ATM signaling to cell cycle checkpoint proteins. The impact of HC is greater than anticipated from the percentage of HC-DNA and, in distinction to DSB repair, ATM only partly overcomes the constraints posed by HC. Importantly, we examine ATM signaling in human syndromes with disordered HC. After depletion of MeCP2 and DNMT3B, proteins defective in the Rett and immunodeficiency with centromere instability and facial anomalies (ICF) syndromes, respectively, we demonstrate enhanced γH2AX signal expansion at HC-chromocenters in mouse NIH 3T3 cells, which have visible HC-chromocenters. Previous studies have shown that the G 2 /M checkpoint is inefficient requiring multiple DSBs to initiate arrest. MeCP2 and DNMT3B depletion leads to hypersensitive radiation-induced G 2 /M checkpoint arrest despite normal DSB repair. Cell lines from Rett, ICF, and Hutchinson-Guildford progeria syndrome patients similarly showed hyperactivated ATM signaling and hypersensitive and prolonged G 2 /M checkpoint arrest. Collectively, these findings reveal that heterochromatin contributes to the previously described inefficient G 2 /M checkpoint arrest and demonstrate how the signaling response can be uncoupled from DSB repair.

DNA double strand break responses and chromatin alterations within the aging cell

“…Given the fact that heterochromatin formation seems to be uncoupled from proliferation arrest, it is unlikely that SAHF are essential for repressing proliferation-associated genes [145]. On the other hand, heterochromatin formation has been shown to suppress DNA damage repair and is consistent with the finding that SAHF are devoid of signaled DSBs [145,157] (Fig. 2C).…”

“…2C). In agreement, human patients suffering from conditions that show disturbed heterochromatin show a hypersensitive and prolonged G2/M checkpoint as well as hyperactivated ATM [157]. However, further investigation is needed to determine how heterochromatin compaction and a decreased DSB signaling are linked to slowly repaired and persistent DSB and the permanent cell cycle arrest during cellular senescence.…”

The Heterochromatic Barrier to DNA Double Strand Break Repair: How to Get the Entry Visa

“…The striking finding was that G2/M checkpoint arrest was activated following exposure to lower doses in Rett Syndrome cells compared to control cells and arrest was maintained for a prolonged period of time. Since DSB repair was normal in Rett Syndrome cells, this suggests that checkpoint arrest was activated and maintained by lower DSB numbers compared to control cell lines (depicted in Figure 3) [33]. A similar finding was observed with ICFa syndrome cells.…”

“…Further, a distinct approach has shown enhanced expansion of γH2AX foci at DSBs within HC relaxed by inhibition, depletion or mutation of defined HC proteins [26,33]. For example, greater γH2AX expansion is observed following addition of HDAC chemical inhibitors [26,34].…”

Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

“…Results represent the MI relative to untreated cells. 1BR hTERT cells shows similar G2/M checkpoint responses to that in 48BR primary cells [40]. The size and number of γH2AX foci clusters between 48BR primary and 1BR hTERT cells were similar (data not shown).…”