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Abstract
Radiotherapy is a ubiquitous mode of cancer treatment which is employed against anarray of malignancies with diverse etiologies. The benefits of radiation, and indeed several
forms of conventional cancer therapy, depend on the formation and persistence of DNA
double strand breaks (DSBs) in cancer cells. Indeed, the ubiquity of radiotherapy illustrates
the fundamental need for cells to maintain genome integrity and repair damaged DNA. While
DSBs form in chromatin by definition, little is known about the influence of epigenetic context
on DSB detection, signaling or repair.
Studies have begun to elucidate the effects of pre-existing, basal variation in chromatin
on DSB repair as well as changes to the epigenome induced after DSBs are detected. Histone
post-translational modifications (PTMs) may differentially impact DSB repair with varying
kinetics. Further, the γH2AX mark formed upon DSB detection is considered a reliable
DSB reporter, but this has not been evaluated genome wide. Indeed, tools to differentiate
DSB induction from DSB repair are lacking, thus impeding efforts to understand how the
epigenome differentially affects stages of the DDR. Here, we introduce new tools which can
separate detection of DSBs from their induction. Using these tools in conjunction with novel
genomic approaches, we uncover links between the basal epigenome and induction and/or
repair of DSBs. We also elucidate mechanisms by which the epigenome is altered following
DSB detection.
Initially, we probed DSB formation and detection genome-wide in the erythroleukemia
cell line K562, taking advantage of ENCODE data to explore the potential influence of local
epigenetic states. Our data revealed non-uniform distributions with respect to chromatin
context; further, DSB induction did not overlap with, γH2AX deposition. In brief, DSBs
in transcribed euchromatin were more readily detected and marked by γH2AX. Next, we
turned to assessing how histone PTMs changed following IR insult. Using proteomics, we
uncovered long-lasting and wide-spread epigenetic alterations. Our data pointed toward
H3K27me3 as a critical regulator of DSB repair. In a third technical development, we
developed a direct DSB labeling method, TdT UdP DSB End Labeling (TUDEL) which
we used to verify changes to the epigenome induced by DSBs. Finally, we uncovered two
mechanisms by which the epigenome impinges upon DSB detection. First, we observed a link
between transcription and DSB repair and validated this using genomic approaches as well
as functional assays. Second, we confirmed previous reports linking the chromatin modifier
PRC2 and its footprint H3K27me3 to the DDR. Extending previous models, we showed that
PRC2 activity post IR is constrained to active euchromatin and link PRC2 and SWI/SNF
activity at DSB loci. These data confirm active recruitment of PRC2 to damaged DNA and
suggest a novel mechanism of action for this chromatin repressor.
Taken together, this work lays out several technical advancements and generates tools
and data sets which will be useful in further analysis of epigenome-DSB interactions. We
revealed novel epigenetic determinants of DSB detection and signaling that may impact
DNA repair and cell survival after irradiation. Several exotic PTMs are implicated in the
DDR through our work and await follow-on studies. Lastly, we refined models of DSB
detection in euchromatin by suggesting that Pol II-dependent transcription mediates rapid
DSB detection, rather than inducing damage. We go on to show that DSBs in repressed
domains and heterochromatin may only be detected during replication. This work identifies
mechanisms that may promote genomic instability and suggests new targets for sensitization
to therapy. In general, we also provide new rationale for cancer-associated epigenetic changes
and suggest that maintenance of genome integrity following IR may be a primary function
of the epigenome and epigenetic modifiers.