Name: Christy Charles
RNA Binding to CBP Stimulates Histone Acetylation and Transcription
Bose, Daniel A., et al
Enhancers are non-coding DNA elements which are associated with transcription activators and chromatin modifiers like CBP and p300. CBP/p300 mediated H3K27 acetylation occurs when the enhancers are active, which is a driving force for the gene transcription. Active enhancers express enhancer RNAs (eRNA) that promote gene expression. Preliminary studies have shown that recruitment of eRNAs and Histone acetyltransferase (HAT) domain of transcription factor p300 lead to gene activation, which is characterized by H3K27 acetylation. How eRNA and CBP/p300 work synergistically to promote gene expression is unknown. We hypothesize that eRNA interacts with CBP and stimulates its HAT activity, thereby facilitating gene expression.
1. Determine the interactions between CBP and eRNA.
We will establish the interaction between RNAs and CBP using cross-linking and immunoprecipitation (IP) techniques. The interacting RNA will be sequenced to ensure that it is eRNA. We will find out whether the interaction between eRNA and CBP is direct or indirect, in case of indirect interaction the mediator proteins involved will be characterized. If adaptor proteins were to be involved their interaction with CBP and eRNA will be established to delineate how they bring together eRNA and CBP.
2. Determine the effect of eRNA-CBP association on CBP mediated H3K27 acetylation.
eRNA-CBP will be pulled down and the acetylation rate of HAT will be assayed. HAT assays will be performed by using varying concentrations of eRNA and HAT domain, the effect of eRNA on the HAT enzyme kinetics will be determined based on the acetylation turnover. The changes in H3K27 acetylation upon eRNA deletion will also be studied. The acetylation activity of CBP is expected to be stimulated by the presence of eRNA.
3. Elucidate the mechanism behind eRNA-CBP mediated gene expression stimulation.
An activation loop blocks the substrate binding site of CBP, which must be displaced to stimulate HAT activity. We will characterize the eRNA interacting domain of CBP and will conceive a model on how eRNA-CBP interaction facilitates activation loop displacement. We anticipate that eRNA will either interact directly with the activation loop or will recruit other factors that will reposition the activation loop and facilitate HAT activation.