Acetylation profiling by Iseq-Kac reveals insights into HSC aging and lineage decision

  • Nat Chem Biol. 2025 May 26. doi: 10.1038/s41589-025-01916-1.
Yanqiu Gong  #  1 ,  Huiwen Zhan  #  1 ,  Ni Wei  #  1 ,  Min Liu  #  1 ,  Yu Liu  #  1 ,  Pengbo Guan  1 ,  Yusi Xie  1 ,  Yujun Deng  1 ,  Qianlun Pu  1 ,  Xiaoxian Lou  1 ,  Xiaodong Wang  1 ,  Rou Zhang  1 ,  Ping Wang  2 ,  Xiuxiu Jin  1 ,  Xiuxuan Wang  1 ,  Zhiqiang Xu  1 ,  Li Gao  1 ,  Xinyuan Wang  1 ,  Siyu He  1 ,  Ying Lu  1 ,  Meng Hu  1 ,  Wanmeng Li  1 ,  Kun Zheng  1 ,  Yong Peng  1 ,  Peng Lei  1 ,  Heng Xu  1 ,  Yujun Shi  3 ,  Jun Qin  4 ,  Hongbo Hu  5  6  7 ,  Huiyuan Zhang  8 ,  Lunzhi Dai  9  10
Affiliations
  • 1. Center for Hematology and Immunology and National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • 2. School of Medical Imaging, Tianjin Medical University, Tianjin, China.
  • 3. Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHC, West China Hospital, Sichuan University, Chengdu, China.
  • 4. State Key Laboratory of Proteomics, Institute of Lifeomics, Beijing, China.
  • 5. Center for Hematology and Immunology and National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China. [email protected].
  • 6. Chongqing International Institute for Immunology, Chongqing, China. [email protected].
  • 7. Tianfu Jincheng Laboratory, Chengdu, China. [email protected].
  • 8. Center for Hematology and Immunology and National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China. [email protected].
  • 9. Center for Hematology and Immunology and National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China. [email protected].
  • 10. Tianfu Jincheng Laboratory, Chengdu, China. [email protected].
  • # Contributed equally.
PMID: 40419771 DOI: 10.1038/s41589-025-01916-1
Abstract

Profiling post-translational modifications face challenges with low-input samples. We developed Iseq-Kac (internal standard-assisted enrichment-free approach for high-throughput quantitative analysis of lysine acetylation) to profile the acetylome in as few as 103-104 cells. By using a hyperacetylated internal standard, Iseq-Kac can be used in mass spectrometry (MS) to enhance MS1 signals and facilitate MS2 fragmentation of acetylated peptides. Using Iseq-Kac, we quantified 675-1,471 acetylated peptides per analysis from 104 hematopoietic stem cells (HSCs) or multipotent progenitors. Validation by targeted MS, site-specific antibodies and functional assays linked aging-related proteome and acetylome changes to HSC lineage decision. A pronounced decrease in acetylation at H4 lysine 77 (H4K77ac) was observed in aged HSCs, linked to histone deacetylase 3 (HDAC3) activity. HDAC3 inhibition or knockdown in HSCs significantly promoted lymphocyte differentiation. Mimicking H4K77ac through H4K77Q expression enhanced B cell differentiation while repressing myeloid differentiation. Overall, Iseq-Kac enables robust low-input acetylome profiling and reveals epigenetic mechanisms underlying lineage skewing in aged HSCs.

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