Research Projects

The chromatin remodeling regulator CECR2 is an epigenetic factor that plays a crucial role in development and chromatin remodeling. CECR2 contains the bromodomain that recognizes acetylated lysine residues in histone and non-histone proteins. CECR2 is known to be upregulated in many cancer types, including breast, ovarian, and lung cancers. However, how CECR2 recruits to chromatin to regulate the expression of CECR2 target genes in various cancers remains unclear. It has been shown that the bromodomain of CECR2 binds weakly to acetylated histone H3K9ac, H3K14ac, and H3K9acK14ac modifications. By contrast, CECR2 has been shown to bind to acetylated non-histone protein RelA through its bromodomain to activate NF-kB target genes and drive breast cancer metastasis. However, there are still limited histone and non-histone interactome available for the CECR2 bromodomain, and this information is crucial to better understand its gene-regulatory functions. This proposal aims to uncover the acetylated histone H4, H3, and H2A and non-histone interacting partners of CECR2 bromodomain and their role in regulating gene expression in various cancers (breast, ovarian, and lung). Our preliminary isothermal titration calorimetry (ITC) binding data suggest that the CECR2 bromodomain strongly recognizes mono acetylated histone H4K5ac, H4K8ac, H4K12ac, and H4K16ac modifications. Next, we plan to explore the interaction of CECR2 bromodomain with different combinations of acetylated histone H4K5acK8ac, H4K5acK12ac, H4K8acK12ac, and H4K5acK8acK12acK16ac modifications. Moreover, the CECR2 and acetylated histone interactions were further validated at the mono nucleosome levels by performing the pull-down assays. Chromatin immunoprecipitation sequencing (ChIP-seq) will be performed to check the localization of CECR2 and acetylated histone H4 marks in the genome. The number of non-histone interacting partners of CECR2 bromodomain could be vast. However, identifying the non-histone interactome of bromodomain is challenging because the affinity of these interactions lies in the micromolar range, which cannot survive harsh treatment during methods like coimmunoprecipitation. To overcome the above challenges, we have developed a powerful chemoproteomic approach for identifying the transient bromodomain-non-histone interactions with temporal precision. In this project we site specifically introduce the photoactivable unnatural amino acid 4-azido-L-phenylalanine (AzF) into the acetyllysine binding pocket of the CECR2 bromodomain. Then the photoactivable CECR2 bromodomain will be utilized to capture the novel acetylated non-histone interacting partners of CECR2 bromodomain from the cellular proteome (cell lysates will be prepared from normal and cancer cells including breast, ovarian, and lung). This data will be crucial for full understanding of CECR2 in transcriptional regulation in multiple cancer types and in the design of small molecule inhibitors for cancer treatment.