Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

Here we utilized the chromatin in vivo assay (CiA) mouse platform to directly examine the epigenetic barriers impeding the activation of the CiA:Oct4 allele in mouse embryonic fibroblasts (MEF)s when stimulated with a transcription factor. The CiA:Oct4 allele contains an engineered EGFP reporter replacing one copy of the Oct4 gene, with an upstream Gal4 array in the promoter that allows recruitment of chromatin modifying machinery. We stimulated gene activation of the CiA:Oct4 allele by binding a transcriptional activator to the Gal4 array. As with cellular reprograming, this process is inefficient with only a small percentage of the cells re-activating CiA:Oct4 after weeks. Epigenetic barriers to gene activation potentially come from heavy DNA methylation, histone deacetylation, chromatin compaction, and other posttranslational marks (PTM) at the differentiated CiA:Oct4 allele in MEFs. Using this platform, we performed a high-throughput chemical screen for compounds that increased the efficiency of activation. We found that Azacytidine and newer generation histone deacetylase (HDAC) inhibitors were the most efficient at facilitating directed transcriptional activation of this allele. We found one hit form our screen, Mocetinostat, improved iPSC generation under transcription factor reprogramming conditions. These results separate individual allele activation from whole cell reprograming and give new insights that will advance tissue engineering.

Considered to be the “guardian of the genome,” the tumor suppressor p53 works to counteract genetic mutation through cell cycle arrest, DNA repair or apoptosis to prevent proliferation of damaged cells. Dysfunction of the p53 pathway is a large driver of tumorigenesis, with an estimated more than 50% of cancers bearing some p53 abnormality; hence, the normalization of p53 function is considered an important clinical goal for cancer therapy. Many of the interactions involving p53 have been extensively studied; however, absent structural information, the identification of the precise nature of the binding interactions proves challenging, more so if they are transient. Using the hydrocarbon stapled peptide methodology previously used by our group to study interactions mediated by alpha-helices, we have begun identifying novel binding targets of the p53 transactivation domain (TAD) as well as its related proteins p63 and p73 through biochemically specific, covalent photochemical crosslinking reactions. Methodologically, we have developed the use of photoreactive stapled alpha helices (pSAHs) representing a unique way to use highly selective, biochemically specific cross-linking reagents. These can be used more broadly to study protein-protein binding interactions in cell-free and cell based assays. The technique has allowed us to learn more about the biologically active state of proteins that interact with the p53-family member TADs as well as the identification of unexplored binding partners and their mechanism of action in disease. Citation Format: Kenneth M. Felsenstein, Kathryn M. Headley, Kevin A. Murray, Elisabeth A. Russell, Federico Bernal. Identification of novel protein modulators of p53 family function using photochemical crosslinking of stapled peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2223. doi:10.1158/1538-7445.AM2013-2223

Mutations in the DNA binding region of tumor suppressor p53 render it transcriptionally inactive (dominant negative) but can also manifest in transcriptionally-independent gain-of-function (GOF) effects, possibly due to mutp53 interacting with other, as yet, unknown targets. Identification of these targets would help to elucidate the pathways and vulnerabilities involved in mutp53 GOF. Covalently linking a known handle to these unknown proteins is one method to aid in their isolation and identification. A hydrocarbon-stapled alpha helical peptide of the p53 transactivation domain (residues 14-29), SAH-p53-8, has been shown to strongly interact with known p53 targets that utilize this domain for binding (HDM2, Kd = 55 nM, HDMX, Kd = 2.3 nM). Substitution of a key interacting residue for the unnatural amino acid benzoylphenylalanine (Bpa) results in a stapled peptide that retains the biochemical properties of SAH-p53-8 (affinities of 38.1 nM and 58.3 nM, respectively) and can covalently capture known targets via photochemical reaction. The introduction of an azido group - a versatile, biochemically-inert capping group - to the N-terminus of this photo-reactive peptide allows for the attachment of various tags to the covalently bound protein(s) via a copper-catazlyzed azide alkyne cycloaddition. Tags include both fluorophores for visualization and affinity tags for protein isolation. This poster will cover the biochemical proof-of-principle of this protein isolation method and exploration into more complex cell lysate environments. Citation Format: Amanda L. Whiting, Joe J. Mitala, Kathryn M. Headley, Joe Reilly, Bethanie L. Morrison, Kevin A. Murray, Federico Bernal. Covalent capture of protein binding partners using an azide-tagged, photo-reactive stapled alpha helical p53 peptide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2014-3234