The majority of cancer patients carry mutations in the p53 gene. Similar to human p53, the Drosophila counterpart is a transcription factor that can respond to genotoxic stress and promote adaptive responses at the cellular level. Our analysis of a Drosophila p53 response element (p53RE) revealed the ability of an enhancer to assemble chromatin conformations to directly activate long distance genes on the same chromosome (cis) as well as in different chromosomes (trans). This mechanism of gene regulation still remains poorly understood and has great impact on the way geneticists study the genome in normal and pathological conditions. Tailored deletion of this p53RE caused visible phenotypes in the fly and prevented stimulus induced gene expression of many p53 target genes in cis and in trans. Chromosome conformation capture (3C) as well as fluorescence in situ hybridization (FISH) indicated that this p53RE regulates its target genes through physical interactions by chromatin looping. In addition, when this single enhancer is reintroduced ectopically to the p53RE flies it restores regulation as well as 3C contacts. To further study the p53RE interaction network, my project's main objectives are: 1) map the genome-wide p53RE interaction profile 2) identify differences in this three dimensional organization in various cell types and 3) determine if the enhancer contacts targets simultaneously or sequentially. To discover new chromatin interaction targets of the p53RE, we will conduct 4C experiments. 4C is a genome-wide chromosome conformation capture assay;it utilizes crosslinking, digestion and intramolecular ligation to identify all in vivo looping contacts to a defined genomic region. Preliminary 4C data from our lab has confirmed previous interaction findings by 3C and reveals additional chromatin interaction networks. To analyze if these chromatin interaction profiles are cell specific and determine if they occur simultaneously we will use both 3C and FISH. Combined, the findings of these studies will examine the capacity of a single enhancer to direct multigenic regulation genome-wide. Furthermore, we will determine if specific contacts organized by enhancers in three dimensions could play a role in cell specific responses.
p53 is a well-recognized tumor suppressor and the most common lesion in human cancer, understanding its method of gene regulation can shed light on the mechanisms cancer cells use to escape this activity.
|D'Brot, A; Kurtz, P; Regan, E et al. (2017) A platform for interrogating cancer-associated p53 alleles. Oncogene 36:286-291|
|Tiwari, Bhavana; Kurtz, Paula; Jones, Amanda E et al. (2017) Retrotransposons Mimic Germ Plasm Determinants to Promote Transgenerational Inheritance. Curr Biol 27:3010-3016.e3|
|Wylie, Annika; Jones, Amanda E; D'Brot, Alejandro et al. (2016) p53 genes function to restrain mobile elements. Genes Dev 30:64-77|
|Link, Nichole; Kurtz, Paula; O'Neal, Melissa et al. (2013) A p53 enhancer region regulates target genes through chromatin conformations in cis and in trans. Genes Dev 27:2433-8|