Capturing the Hierarchically Assorted Modules of Protein Interaction in the Organized Nucleome
Authors: Dai Shuaijian , Liu Shichang , Zhou Chen , Yu Fengchao , Zhu Guang , Zhang Wenhao , Deng Haiteng , Burlingame Al , Yu Weichuan , Wang Tingliang , Li Ning 
Published: August 2022

Nuclear proteins are major constituents and key regulators of the topological organization of nucleome. To elucidate the global connectivity of nucleomic proteins and to decipher the hierarchically organized modules of protein interaction that are involved in nucleomic organization and nuclear events, both formaldehyde and CBDPS crosslinkers were applied sequentially on the in vivo prefixed nuclei to perform a double chemical crosslinking with mass spectrometry (XL-MS) analysis. The integration of dimethyl-labelling with XL-MS generated a quantitative XL-MS workflow (qXL-MS) that consequently identified 5,340 cross-linked peptides (crosslinks) from nucleome. These crosslinks were construed into 1,297 nuclear protein-protein interactions (PPIs), from which discovered were 250 and 26 novel interactors of histones and nucleolar box C/D snoRNP complex, respectively. MONET-based modulomic analysis of their Arabidopsis orthoglous PPIs constructed 27 and 24 master nuclear protein interaction modules (NPIMs) that contain the condensate-forming protein(s) and the intrinsically disordered region (IDR)-containing proteins, respectively. These NPIMs successfully captured the previously reported nuclear protein complexes and nuclear bodies in nucleome. Surprisingly, modulomic analysis showed that these NPIMs were hierarchically assorted into four communities of NPIMs in nucleome including Genome Community and Nucleolus Community. The qXL-MS-based quantitative interactomics finally revealed 17 Hormone-specific module variants participating in a broad range of nuclear events. Thus, this integrated pipeline of qXL-MS and MONET modulomics, named as CHAMPION, is capable of capturing both nuclear protein complexes and nuclear bodies, constructing the topological architecture of protein interaction modules and module variants in nucleome and probably of mapping the protein compositions of condensates.

Highlights

The formaldehyde and CBDPS crosslinkers coupled qXL-MS discovered 5,340 crosslinked peptides. These crosslinks were construed into 1,297 nuclear protein-protein interactions (PPIs), protein components of which contained 250 and 26 novel interactors of histone octamer and nucleolar box C/D snoRNP complex, respectively, in the intricately organized nucleome.

The MONET-based modulomic analysis of these crosslinks captured 95 nuclear protein interaction modules (NPIMs), a portion of which contain both the condensate-forming and the intrinsically disordered region (IDR)-containing proteins. Especially, some NPIMs captured 6 previously reported nuclear protein complexes.

A number of Hormone-specific module variants were identified by modulomics upon hormone treatment using the hormone significantly up-regulated crosslinks from qXL-MS. Several PPIs and NPIMs have been substantiated with alternative biological experiments.

This CHAMPION pipeline has partitioned these NPIMs into four hierarchically and topologically organized communities in nucleome. The molecular functions of those proteins partitioned into C1 and C2 community are specialized in genome organization and nucleolar functions, respectively.