Mus musculus Epigenomics

Current methods for profiling post-translational modifications (PTMs) face challenges with low-input samples. Here we developed Iseq-Kac (internal standard-assisted enrichment-free approach for high-throughput quantitative analysis of lysine acetylation), a technique that enables acetylomic profiling of as few as 103-104 cells. This method employs a chemically acetylated internal standard to enhance MS1 signals of acetylated peptides and facilitate MS2 fragmentation. Using Iseq-Kac, we profiled the aging-related proteome and acetylome of hematopoietic stem and progenitor cells (HSPCs) from 104 cells, quantifying an average of 675 to 1,471 acetylated peptides from hematopoietic stem cells (HSCs) or multipotent progenitors (MPPs) per analysis. These findings were extensively validated through targeted mass spectrometry analysis, site-specific antibodies, and functional assays. Aging-related proteome and acetylome of HSPCs are linked to metabolic regulation and HSC lineage decision. As chromatin senses cellular metabolism through histone modifications, we focused on histone acetylation. Notably, we observed a substantial decrease in histone mark H4K77ac in aged HSCs. We identified HDAC3 as the deacetylase of H4K77ac, and its inhibition or knockdown in HSCs significantly promoted lymphocyte differentiation. Further mimicking H4K77ac through ectopic expression of H4K77Q also enhanced in vivo B cell differentiation while repressing myeloid differentiation. Overall, this work presents a cutting-edge technique for acetylomic analysis of low-input samples and highlights the epigenetic regulation of lineage skewing in aged HSCs, which may help rebalance lineage output and rescue myeloid skewing.