Genome-wide mapping reveals that deoxyuridine is enriched in the human centromeric DNA.

Uracil in DNA can be generated by cytosine deamination or dUMP misincorporation; however, its distribution in the human genome is poorly understood. Here we present a selective labeling and pull-down technology for genome-wide uracil profiling and identify thousands of uracil peaks in three different human cell lines. Surprisingly, uracil is highly enriched at the centromere of the human genome. Using mass spectrometry, we demonstrate that human centromeric DNA contains a higher level of uracil. We also directly verify the presence of uracil within two centromeric uracil peaks on chromosomes 6 and 11. Moreover, centromeric uracil is preferentially localized within the binding regions of the centromere-specific histone CENP-A and can be excised by human uracil-DNA glycosylase UNG. Collectively, our approaches allow comprehensive analysis of uracil in the human genome and provide robust tools for mapping and future functional studies of uracil in DNA.

Transcriptome-wide mapping reveals reversible and dynamic N(1)-methyladenosine methylome.

N(1)-Methyladenosine (m(1)A) is a prevalent post-transcriptional RNA modification, yet little is known about its abundance, topology and dynamics in mRNA. Here, we show that m(1)A is prevalent in Homo sapiens mRNA, which shows an m(1)A/A ratio of ∼0.02%. We develop the m(1)A-ID-seq technique, based on m(1)A immunoprecipitation and the inherent ability of m(1)A to stall reverse transcription, as a means for transcriptome-wide m(1)A profiling. m(1)A-ID-seq identifies 901 m(1)A peaks (from 600 genes) in mRNA and noncoding RNA and reveals a prominent feature, enrichment in the 5' untranslated region of mRNA transcripts, that is distinct from the pattern for N(6)-methyladenosine, the most abundant internal mammalian mRNA modification. Moreover, m(1)A in mRNA is reversible by ALKBH3, a known DNA/RNA demethylase. Lastly, we show that m(1)A methylation responds dynamically to stimuli, and we identify hundreds of stress-induced m(1)A sites. Collectively, our approaches allow comprehensive analysis of m(1)A modification and provide tools for functional studies of potential epigenetic regulation via the reversible and dynamic m(1)A methylation.

Base-Resolution Analysis of Cisplatin-DNA Adducts at the Genome Scale.

Cisplatin, one of the most widely used anticancer drugs, crosslinks DNA and ultimately induces cell death. However, the genomic pattern of cisplatin-DNA adducts has remained unknown owing to the lack of a reliable and sensitive genome-wide method. Herein we present "cisplatin-seq" to identify genome-wide cisplatin crosslinking sites at base resolution. Cisplatin-seq reveals that mitochondrial DNA is a preferred target of cisplatin. For nuclear genomes, cisplatin-DNA adducts are enriched within promoters and regions harboring transcription termination sites. While the density of GG dinucleotides determines the initial crosslinking of cisplatin, binding of proteins to the genome largely contributes to the accumulative pattern of cisplatin-DNA adducts.

Therapeutic delivery of siRNA silencing HIF-1 alpha with micellar nanoparticles inhibits hypoxic tumor growth.

The particular characteristics of the tumor microenvironment have the potential to strongly promote tumor growth, metastasis and angiogenesis and induce drug resistance. Therefore, the development of effective, systemic therapeutic approaches specifically based on the tumor microenvironment is highly desirable. Hypoxia-inducible factor-1α (HIF-1α) is an attractive therapeutic target because it is a key transcription factor in tumor development and only accumulates in hypoxic tumors. We report here that a cationic mixed micellar nanoparticle (MNP) consisting of amphiphilic block copolymers poly(ε-caprolactone)-block-poly(2-aminoethylethylene phosphate) (PCL(29)-b-PPEEA(21)) and poly(ε-caprolactone)-block-poly(ethylene glycol) (PCL(40)-b-PEG(45)) was a suitable carrier for HIF-1α siRNA to treat hypoxic tumors, which showed an average diameter of 58.0 ± 3.4 nm. The complex MNP(siRNA), formed by the interaction of MNP and siRNA, was transfected into PC3 prostate cancer cells efficiently, while the inhibition of HIF-1α expression by MNP loaded with HIF-1α siRNA (MNP(siHIF)) blocked PC3 cell proliferation, suppressed cell migration and disturbed angiogenesis under in vitro hypoxic mimicking conditions. It was further demonstrated that systemic delivery of MNP(siHIF) effectively inhibited tumor growth in a PC3 prostate cancer xenograft murine model without activating innate immune responses. Moreover, delivery of MNP(siHIF) sensitized PC3 tumor cells to doxorubicin chemotherapy in vitro and in vivo by downregulating MDR1 gene expression which was induced by hypoxia. The underlying concept of use of MNP(siHIF) to block HIF-1α holds promise as an example of a clinical approach using specific siRNA therapy for cancer treatment aimed at the hypoxic tumor microenvironment.