Polyphosphate Modification of Human Proteins and a New Mechanism for Regulating Super-Enhancer Activity

Inorganic polyphosphate (polyP) is an evolutionarily conserved linear polymer of orthophosphate that plays diverse regulatory roles across biological systems. Recent work has implicated that polyP modulates protein function through post-translational modification (PTM), which has gained increasing scientific interest. However, its human protein interactome and molecular functions especially its role in gene regulation in human cells remain poorly defined. This thesis demonstrated that polyP serves as a multifaceted regulatory molecule in human cellular biology, identified many human proteins containing lysine-rich regions as polyP targets and uncovered polyP’s role in manipulating super-enhancer complex to regulate gene expression. Through a comprehensive screen of 57 lysine-rich human proteins, selected via mass spectrometry and bioinformatic analyses, this study identified 41 proteins that undergo denaturation-resistant polyP modification. Through mutagenesis and binding assays, this study established that intrinsically disordered lysine-rich motifs are essential for polyP modification, with consecutive lysine residues as critical determinants. Functional assays further revealed that polyP binding modulated the activity of diverse regulatory proteins - it inhibited phase separation of the transcriptional regulator NKAP, suppressed GTPase activation of the oncogenic driver KRAS, and reduced the helicase activity of DDX55. Building on this framework, this study also identified a previously unrecognized role for polyP in transcriptional regulation through its direct interaction with components of the mammalian super-enhancer complex. PolyP modified the Mediator subunit MED1, the coactivator BRD4, and the transcription factor YY1. These modifications disrupted their capacity for phase separation, reduced MED1 and BRD4 expression and impaired YY1 nuclear localization. In addition, polyP inhibited YY1 dimer formation and disrupted YY1-mediated DNA looping, ultimately attenuating super-enhancer driven transcription. Collectively, this thesis provides a comprehensive resource for polyP studies and unveils a novel gene regulation role of polyP, exploring the physiological and pathological significance of polyP while establishing it as an important player to epigenetic regulation, cellular homeostasis, and human disease.

Department dc:contributor.department

Biomedical and Molecular Sciences

Year dc:date.issued

2026

Author dc:creator

• Yang, Zhiyun

Advisor dc:contributor.supervisor

• Jia, Zongchao

• Polyphosphate
• Lysine-rich proteins
• Gene regulation
• Super-enhancers
• PolyP modification
• PolyP function

Statement dc:rights

• Attribution-NonCommercial-ShareAlike 4.0 International

Licence dc:rights.uri

• http://creativecommons.org/licenses/by-nc-sa/4.0/

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1974/36408

OAI identifier oai:identifier

oai:queensu.scholaris.ca:1974/36408