Deciphering the molecular mechanisms of histone code recognition by ATAD2/B

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ATAD2 is an important co-activator of the estrogen and androgen receptors2,4. ATAD2 is known to be up- regulated in multiple different types of cancer including breast, lung, gastric, endometrial, renal, and prostate5,7,9-12. Up-regulation of ATAD2 is often correlated with poor patient outcomes, and can be used as prognostic marker9,12-14. Furthermore, silencing the expression of ATAD2 through RNA interference inhibits cell proliferation and promotes apoptosis in ovarian carcinoma, and inhibits migration and invasion of hepatocellular carcinoma and colorectal cancer cells13,15,16. ATAD2B, is a poorly studied paralog of the ATAD2 gene, and although ATAD2 and ATAD2B are highly conserved, there is little known about the function of ATAD2B or its role in oncogenesis. Both the ATAD2/B proteins contain two conserved domains: an AAA ATPase domain and a bromodomain6. The overall objective of the proposed research is to determine how di- acetyllysine recognition by the ATAD2/B bromodomains regulates the cellular function of these proteins. This proposal aims to: (1) characterize how cross-talk between histone modifications modulate acetyllysine recognition by the ATAD2/B bromodomains; (2) outline the molecular mechanism(s) of di-acetylated histone recognition by the ATAD2/B bromodomains; (3) determine the functional significance of di-acetyllysine recognition by the ATAD2/B bromodomains. A unique combination of in vitro biochemical, biophysical, and structural biology studies on the ATAD2/B bromodomains will be coupled with in vivo functional genomic investigations using a breast cancer progression model to characterize the biological roles of the ATAD2/B bromodomains. We will evaluate the impact of neighboring histone modifications on histone H4 tail recognition using peptide array assays in combination with isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) chemical shift perturbation techniques. The atomic resolution structures of the ATAD2/B bromodomains bound to their di-acetylated histone tail ligands will be solved by NMR or X-ray crystallography. To characterize the binding mode of the ATAD2/B bromodomains with their histone ligands we will carry out analytical ultracentrifugation, size-exclusion chromatography, ITC, and NMR T1/T2 relaxation experiments. Site-directed mutagenesis coupled with NMR and ITC will be used to measure the effects on ligand binding, and identify differences in the binding pockets of the ATAD2/B bromodomains. We will compare the genome- wide associations of ATAD2/B with histone H4 acetylation patterns in a breast cancer progression model to determine the impact of ATAD2/B on breast cancer cell phenotypes using ChIP-seq and RNA-seq, followed by cellular migration and invasion assays. Our multi-faceted approach will correlate specific histone modifications with ATAD2/B binding and action, which will allow us to connect histone H4 acetylation marks to bromodomain function in cancer cell proliferation. Overall, our integrated biochemical, biophysical, structural biology and functional genomics approach will facilitate the discovery of novel drug targets to help overcome cancer.