Transcription restores DNA repair to heterochromatin, determining regional mutation rates in cancer genomes

Abstract

Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC(-/-) background. XPC(-/-) cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.

Figure 1. Regional disparities in mutation density are absent in non-expressed portions of the genome of germline XPC^−/− squamous cell carcinomas

The x-axis of each graph shows increasing ChIP intensity of the heterochromatin-associated histone mark H3K9me3 (ENCODE data, Broad Institute, Panels A, C) and increasing inverse median RepliSeq values representing later replication time (ENCODE data, University of Washington, Panels B, D). The y-axis represents the mutation density per Kb divided by the individual mean. Plotted are values for either 8 aggregated repair wild-type (WT) cancers (solid blue line) or 5 aggregated XPC^−/− cancers (broken orange line) for 8 equally sized genomic bins covering approximately 2Gb of expressed genome and 1Gb of non-expressed genome (+/− STD). Whereas mutation density correlates positively with increasing H3K9me3 and later replication time for expressed regions in repair wild-type cancers, these associations are diminished in XPC^−/− samples (Panels A, B). In non-expressed portions of the genome, regional disparities in mutation density are almost completely abolished in XPC^−/− samples (Panels C, D), indicating loss in the absence of GG-NER. See Figure S1 for additional data with sparser active marks H3K27ac and H3K4me1 and Table S1 for additional information on tumor samples.

Figure 2. Domain-associated repair restores low mutation rate only to highly transcribed genes in tightly-packaged DNA

The x-axis denotes increasing expression in NHEK, measured in RPKM (plotted on a log scale). On the y-axis is the mutation density per Kb. Values are plotted for three independent WT cSCCs (A–C) and three independent XPC^−/− cSCCs (D–F). The plots show six different H3K9me3 densities representing different chromatin levels, represented by distinct colors, for the transcribed (solid line) and untranscribed (broken line) strands. In WT cancers, both strands show decreasing mutation density in tightly-packaged DNA, illustrating robust domain-associated repair (DAR). DAR restores mutation rate in the most heterochromatic genomic regions to that of euchromatic regions, evidencing a dominant effect over chromatin state, but negligible additional impact in euchromatin (low H3K9me3). Even lower mutation density is seen from lesions on the transcribed strand, presumably representing TC-NER. In contrast, the XPC^/− cancers show an absence of DAR, represented by an absence of transcription-dependent repair on the untranscribed strand, but intact TC-NER. See Figure S2 for additional samples and Table S2–S6 for more detailed mutation density information.

Figure 3. Gene expression significantly alters tumor suppressor mutation rates

The x-axis shows increasing H3K9me3 intensity, representing a more repressive chromatin state. The y-axis shows the fold increase of the probability of a mutation, given a 50% decrease in expression level, referred to here as θ. Plotted is θ for 20,841 1Kb segments covering transcribed portions of 261 genes recently identified as recurrently mutated in human cancers. Highlighted are 1Kb fragments containing exons for the SCC tumor suppressors TP53 (A), NOTCH1 (B), IRF6 (C), as well as for the gene with exons of greatest average level of such mutation variance, CDC27 (D), which has been shown to be mutated at about 4% in melanomas and 2% in head and neck SCCs. Exons with the highest variance and its corresponding θ are indicated. See Table S7 for θ for all 20,841 1Kb segments.