Epigenomic profiling by chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is a prevailing methodology used to investigate chromatin-based regulation in biological systems such as human disease, but the lack of an empirical methodology to enable normalization among experiments has limited the precision and usefulness of this technique. Here, we describe a method called ChIP with reference exogenous genome (ChIP-Rx) that allows one to perform genome-wide quantitative comparisons of histone modification status across cell populations using defined quantities of a reference epigenome. ChIP-Rx enables the discovery and quantification of dynamic epigenomic profiles across mammalian cells that would otherwise remain hidden using traditional normalization methods. We demonstrate the utility of this method for measuring epigenomic changes following chemical perturbations and show how reference normalization of ChIP-seq experiments enables the discovery of disease-relevant changes in histone modification occupancy.

SUMMARY Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or β (PI5P4Kα and β) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and β in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K β and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A−/−, PIP4K2B+/−, and TP53−/− mice were viable and had a dramatic reduction in tumor formation compared to TP53−/− littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.

Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to .1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research. C ONVENTIONAL gene targeting technology in mice relies on homologous recombination in embryonic stem (ES) cells to target specific gene sequences, most commonly to disrupt gene function (Doetschman et al. 1987;Kuehn et al. 1987;Thomas and Capecchi 1987). Advantages of gene targeting in ES cells are selective target sequence modification, the ability to insert or delete genetic information, and the stability of the targeted mutations through subsequent generations. There are also potential limitations, including limited rates of germline transmission and strain limitations due to lack of conventional ES cell lines (Ledermann 2000;Mishina and Sakimura 2007). Moving the targeted allele from one strain to another requires 10 generations of backcrosses that take 2-3 years. A minimum of 1 year is necessary for backcrossing if speed congenics is applied (Markel et al. 1997).Zinc-finger nucleases (ZFNs) are fusions of specific DNA-binding zinc finger proteins (ZFPs) and a nuclease domain, such as the DNA cleavage domain of a type II endonuclease, FokI (Kim et al. 1996;Smith et al. 1999;Bibikova et al. 2001). A pair of ZFPs provide target specificity, and their nuclease domains dimerize to cleave the DNA, generating double strand breaks (DSBs) (Mani et al. 2005), which are detrimental to the cell if left unrepaired (Rich et al. 2000). The cell uses two main pathways to repair DSBs: high-fidelity homologous recombination and error-prone nonhomologous end joining (NHEJ) (Lieber 1999;Pardo et al. 2009;Huertas 2010). ZFN-mediated gene disruption results from deletions or insertions frequently introduced by NHEJ. Figure 1 illustrates the cellular events following the injec...