Molecular mechanism of UV damage modulation in nucleosomes.

Exposure to ultraviolet (UV) light causes the formation of mutagenic cyclobutane pyrimidine dimers (CPDs) in cellular DNA. Previous studies have revealed that CPD formation in nucleosomes, the building blocks of chromatin, shows a striking ∼10 base pair (bp) periodic pattern. CPD formation is suppressed at positions where the DNA minor groove faces toward the histone octamer (minor-in) and elevated CPD formation at positions where the minor groove faces away from the histone octamer (minor-out). However, the molecular mechanism underlying this nucleosome photofootprint is unclear. Here, we analyzed ∼180 high-resolution nucleosome structures to characterize whether differences in DNA mobility or conformation are responsible for the CPD modulation in nucleosomes. Our results indicate that differences in DNA mobility cannot explain CPD modulation in nucleosome. Instead, we find that the sharp DNA bending around the histone octamer results in DNA conformations with structural parameters more susceptible to UV damage formation at minor-out positions and more resistant to CPD formation at minor-in positions. This analysis reveals the molecular mechanism responsible for periodic modulation of CPD formation and UV mutagenesis in nucleosomal DNA.

CTCF binding modulates UV damage formation to promote mutation hot spots in melanoma.

Somatic mutations in DNA-binding sites for CCCTC-binding factor (CTCF) are significantly elevated in many cancers. Prior analysis has suggested that elevated mutation rates at CTCF-binding sites in skin cancers are a consequence of the CTCF-cohesin complex inhibiting repair of UV damage. Here, we show that CTCF binding modulates the formation of UV damage to induce mutation hot spots. Analysis of genome-wide CPD-seq data in UV-irradiated human cells indicates that formation of UV-induced cyclobutane pyrimidine dimers (CPDs) is primarily suppressed by CTCF binding but elevated at specific locations within the CTCF motif. Locations of CPD hot spots in the CTCF-binding motif coincide with mutation hot spots in melanoma. A similar pattern of damage formation is observed at CTCF-binding sites in vitro, indicating that UV damage modulation is a direct consequence of CTCF binding. We show that CTCF interacts with binding sites containing UV damage and inhibits repair by a model repair enzyme in vitro. Structural analysis and molecular dynamic simulations reveal the molecular mechanism for how CTCF binding modulates CPD formation.

CTCF puts a new twist on UV damage and repair in skin cancer.

Somatic mutations in skin cancers are highly enriched at binding sites for CCCTC-binding factor (CTCF). We have discovered that CTCF binding alters the DNA structure to render it more susceptible to UV damage. Elevated UV damage formation at CTCF binding sites, in conjunction with subsequent repair inhibition, promotes UV mutagenesis.

Mapping diet-induced alternative polyadenylation of hypothalamic transcripts in the obese rat.

RNA biogenesis has emerged as a powerful biological event that regulates energy homeostasis. In this context insertion of alternative polyadenylation sites (APSs) dictate the fate of newly synthesized RNA molecules and direct alternative splicing of nascent transcripts. Thus APSs serve a mechanistic function by regulating transcriptome expression and function. In this study we employed a novel RNA-Seq Next Generation Sequencing (NGS) approach that utilized the power of Whole Transcriptome Termini Site Sequencing (WTTS-Seq) to simultaneously measure APS events on multiple RNA biotypes. We used this technique to measure APS events in the hypothalamus of adult male Long Evans rats exposed to a palatable high fat diet (HFD) or chow. Rats maintained on HFD displayed typical hyperphagic feeding and ensuing body weight gain over the one-month manipulation period. Our WTTS-Seq analysis mapped approximately 89,000 unique hypothalamic APSs induced by HFD relative to chow fed controls. HFD exposure produced APSs on multiple RNA biotypes in the hypothalamus. The majority of detected APSs occur on mRNA transcripts that encode functional proteins. Notably we find APSs on micro (miRNA) and long non-coding RNAs (lncRNA), newly recognized transcription factors that regulate body weight in rodents. In addition we detect APSs on protein encoding mRNAs that control neuron projection development and synapse organization and glutamate signaling, key events hypothesized to maintain excess food intake. Importantly, quantitative real time PCR indicated that APS insertion led to increased hypothalamic expression of multiple RNA biotypes. Collectively these data highlight APS events as a novel genetic mechanism that directs hypothalamic RNA biogenesis stimulated by diet-induced obesity.