Interrogating chromatin-associated proteins with multifunctional nucleosome-based chemical probes

Abstract

Genomic information of eukaryotic cells is stored in a DNA-protein complex termed chromatin. The basic repeating unit of chromatin is nucleosome, which consists of ~146 base pairs of DNA wrapping around a histone octamer containing two copies of histone H2A, H2B, H3 and H4 respectively. Besides DNA and histone per se, the composition and organization of chromatin are also orchestrated by many chromatin-associated proteins, such as the enzymes that add or remove histone posttranslational modifications (PTMs), the effector proteins that “read” and translate histone PTMs and chromatin remodelers that modulate nucleosome assembly. The landscape of chromatin determines the accessibility of underlying DNA, thus affecting many DNA-templated processes such as gene transcription, DNA replication and damage repair. Malfunction of chromatin-associated proteins often results in dysregulation of these fundamental cellular processes and leads to human diseases such as cancer. Therefore, it is essential to carefully interrogate chromatin-associated proteins. Current methods to study chromatin-mediated protein interactions (summarized in chapter 1) either have difficulty in identifying direct interactors and characterizing the specific structural elements that mediate the interactions, or fail to profile the nucleosome context-dependent interactions. Furthermore, the proteins are usually involved in weak and transient interactions with chromatin, thus are easily lost during biochemical pulldown processes. To address these problems, we developed the multifunctional nucleosome-based chemical probes that are site-specifically equipped with (i) a photo-reactive group to covalently trap weak interactions upon UV irradiation; (ii) a bioorthogonal handle facilitating the isolation of crosslinked proteins; (iii) a disulfide linkage that can rapidly release crosslinked peptide for mass spectrometry (MS) analysis. When coupled with state-of-art MS, the nucleosome-based probes can not only identify specific chromatin-associated proteins from complex proteomes, but also characterize the binding regions of proteins that bind to chromatin in a nucleosomal context. In chapter 2, two cysteine-reactive molecules I-Dzyne and Npys-Dzyne are introduced for efficient incorporation of multifunctional moieties to “bait” probes. The multifunctionality was validated by the derived peptide- and protein-based probes in capturing known interactors and mapping the binding site. In chapter 3, nucleosome-based probes were generated to scrutinize the association of H3K79-methyltransferase Dot1L, which binds to chromatin in a context-dependent manner. The probes can not only identify the binding site, but also serve as distance sensors to semi-quantitatively interrogate the Dot1L-nucleosome interaction. In chapter 4, chemically synthesized multifunctional nucleosome probes were used to profile H3K79me2-specific binders. As a result, protein Menin was identified and biochemically validated to be a “reader” that specifically recognize di-methylated H3K79 in a nucleosome-dependent manner.