Histone Variant Regulates DNA Repair via Chromatin Condensation
DNA break-induced chromatin condensation promotes BRCA1-dependent genome maintenance.
Activating the appropriate DNA repair pathway is essential for maintaining the stability of the genome after a break in both strands of DNA. How a pathway is selected, however, is not well understood. Since these double strand breaks (DSBs) occur while DNA is packaged as chromatin, changes in its organization are necessary for repair to take place. Numerous alterations have been associated with DSBs, including modifications of histone tails and exchange of histone variants, some increasing chromatin accessibility, others reducing it. In fact, distinct domains flanking a single DSB have been observed that are bound by opposing repair pathway proteins 53BP1and BRCA1, which promote non-homologous end joining (NHEJ) and homologous recombination (HR), respectively. To investigate whether DSB-proximal chromatin reorganization affects repair pathway selection, Philipp Oberdoerffer, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues performed a high-throughput RNA interference (RNAi) screen for chromatin-related genes that modulate HR.
The researchers identified the repressive histone variant macroH2A1 as one of the top-five HR promoting candidates. The macroH2A1-encoding gene, H2AFY, actually produces two splice variants, macroH2A1.1 and macroH2A1.2, the latter lacking a carboxy-terminal poly-ADP-ribose (PAR) binding domain. To tease apart the variants’ roles, the investigators knocked down macroH2A1.2 or both isoforms and generated DSBs by inducing the expression of endonuclease I-SceI. They observed reduced HR efficiency that correlated with the reduction in macroH2A1.2, suggesting that macroH2A1, particularly variant 1.2, facilitates HR. Using chromatin immunoprecipitation, the scientists demonstrated that macroH2A1 accumulates at DSB sites along with known repair markers. Laser microirradiation and immunostaining allowed the researchers to monitor the kinetics of macroH2A1.2 recruitment and revealed an initial depletion from DSBs followed by re-accumulation within minutes and prolonged enrichment.
Since macroH2A1 is associated with chromatin silencing, the investigators examined whether other repressive marks coincided with its accumulation. They found that dimethylation of histone H3 lysine 9 (H3K9me2) was also enriched following nuclease- and laser-induced DSBs and with the same kinetics as macroH2A1. Knocking down macroH2A1 or variant 1.2 reduced H3K9me2 at DSBs, suggesting the two modifications are linked. As macroH2A1 has no methyltransferase activity, the scientists reexamined their RNAi screen data for these enzymes and identified PRDM2, a known modifier of H3K9 that had not been implicated previously in DSB repair.
Reducing the expression of PRDM2 decreased HR efficiency and reduced H3K9me2 after DSB induction. The researchers then showed that PRDM2 was recruited to DSBs just after H3K9me2 depletion and remained enriched at these sites. Similar to H3K9me2, knocking down macroH2A1 or variant 1.2 impaired PRDM2 accumulation at DSBs. In contrast, loss of PRDM2 had no effect on macroH2A1 recruitment. Knocking down both macroH2A1 and PRDM2 had no additive effect on HR efficiency, suggesting they act in the same DSB repair pathway. Because chromatin changes are linked with activation of the DNA damage signaling protein ATM, the investigators examined its involvement in their newly-identified pathway. In the presence of an ATM inhibitor, they observed prolonged depletion and reduced accumulation of macroH2A1 at DSBs as well as impaired PRDM2 recruitment and H3K9me2, demonstrating that the macroH2A1/PRDM2 pathway depends on ATM signaling.
The scientists next evaluated the functional consequences of macroH2A1 accumulation at DSBs. Using a nuclease-based assay, they found reduced nuclease sensitivity near DSBs in control cells but saw increased chromatin accessibility and reduced H3K9me2 when macroH2A1 was knocked down, suggesting that initial DSB-induced chromatin relaxation was followed by macroH2A1-dependent condensation. To examine this effect over time in living cells, the researchers used a cell line expressing a photoactivatable green fluorescent protein fused to histone H2B. Immediately after laser-induced DSB formation, they observed rapid chromatin expansion. DSB flanking regions then recondensed within minutes, coinciding with maximal PRDM2 accumulation. Loss of either macroH2A1 or PRDM2 reduced chromatin compaction but not expansion. Inhibition of ATM also impaired condensation, while blocking PAR polymerase (PARP) blunted chromatin expansion. These results demonstrate that the two phases of DSB-proximal chromatin reorganization are regulated by distinct pathways with condensation dependent on the repressive components macroH2A1 and PRDM2.
Finally, the investigators examined the link between loss of these regulators of chromatin condensation and defects in HR. Previous studies indicated that BRCA1 accumulates at condensed chromatin. The scientists found that knocking down macroH2A1, variant 1.2, or PRDM2 impaired BRCA1 recruitment to DSBs, the timing of which coincided with macroH2A1/PRDM2-mediated chromatin condensation. 53BP1, however, showed no change in accumulation at DSBs, indicating that the effect was specific for BRCA1 and suggesting that macroH2A1 and PRDM2 may play a role in repair pathway choice. In fact, loss of macroH2A1.2 or PRDM2 significantly reduced HR with no change or a slight upregulation in NHEJ; reducing the expression of 53BP1 partially restored these HR defects. 53BP1 is known to antagonize HR in cells lacking BRCA1 by interfering with DNA end resection by CtIP. Consistent with this, the researchers found a reduction in CtIP recruitment to DSBs in cells lacking macroH2A1.2 or PRDM2. Also analogous to BRCA1-deficient cells, knocking down macroH2A1.2 or PRDM2 increased sensitivity to PARP inhibition, which was partially rescued by 53BP1 loss.
Together these studies demonstrate that macroH2A1 and PRDM2 guide DSB repair via HR by promoting chromatin condensation, BRCA1 recruitment, and CtIP-mediated DNA end resection. Based on the importance of this pathway in cancer, macroH2A1, and PRDM2 may represent novel therapeutic targets.Summary Posted: Mon, 09/01/2014
Khurana S, Kruhlak MJ, Kim J, Tran AD, Liu J, Nyswaner K, Shi L, Jailwala P, Sung MH, Hakim O, Oberdoerffer P. A Macrohistone Variant Links Dynamic Chromatin Compaction to BRCA1-Dependent Genome Maintenance. Cell Rep. August 21, 2014 PubMed Link