Differences in transcriptional regulation contribute tremendously to phenotypic diversity, including susceptibility to human disease. Transcription is regulated by the sequence-specific binding of transcription factors (TFs) to DNA, typically within promoter and enhancer regions, where TF binding often correlates with the expression of bidirectional transcripts. Work in our lab suggests that these transcripts represent a highly informative signature of TF activity, and that eRNAs may be directly regulated by TF binding.
Aim 1 seeks to determine whether the binding of a single TF, p53, can cause eRNA transcription. We will initially focus on a single locus to determine whether p53 binding alone can initiate eRNA expression. The DRAM1 gene, as well as a p53-dependent eRNA about 20kb upstream, is rapidly induced upon treatment with the p53-activating drug, Nutlin-3. I will use CRISPR-Cas9 to disrupt the p53 binding site within the DRAM1 eRNA in order to prevent p53 from binding following p53 stimulation. We will use nuclear run on RT-qPCR to determine whether the loss of p53 binding results in the loss of DRAM1 eRNA and/or gene transcription. Next, we will attempt to rescue p53 binding at this locus by tethering the p53 transactivation domain to dCas9 and guiding the construct to the mutated p53 binding site and testing whether eRNA transcription is restored. This innovative approach will prove definitively whether the binding of a single TF can cause eRNA transcription, and whether eRNA transcription is correlated with increased gene expression. Missense mutations within TFs are common in cancer, but their functional impact is often difficult to predict. Several p53 missense mutations commonly observed in cancer alter p53s DNA binding specificity and target gene expression, and can lead to increased cellular proliferation and poor prognosis relative to complete loss of p53 function. Such mutations often occur within the p53 DNA binding domain, resulting in altered P53 transactivating capacity and modified physical interactions between p53 and other TFs. To determine whether p53 mutants display altered p53 binding and/or eRNA expression profiles, we will use CRISPR-Cas9 to generate cell lines expressing a single copy of mutant and wildtype forms of p53 in HCT116 p53-/- cells and perform p53 ChIP-seq and GRO-seq before, and one hour after p53 activation by nutlin. From these analyses, we will recover direct p53 wildtype- and mutant- specific gene and eRNA targets. Next, we will perform enhancer profiling to test whether other TFs are activated in the presence of p53 mutants. Finally, we will ask whether drugs designed to recover wildtype p53 function in mutant p53 strains are capable of restoring p53 wildtype eRNA and gene expression profiles.
Transcription factors are major determinants of cell type and state. However, no reliable methods for quantifying TF activity on a global scale exist to date. We developed an approach, enhancer profiling, that can simultaneously infer the activity of each TF throughout the genome and at individual loci from a single experiement. By combining our approach with state of the art molecular biology tools, we seek to improve our fundamental knowledge of transcriptional regulation and its dysregulation in human disease.
|Azofeifa, Joseph G; Allen, Mary A; Hendrix, Josephina R et al. (2018) Enhancer RNA profiling predicts transcription factor activity. Genome Res :|