Normal adjacent tissues (NATs) of hepatocellular carcinoma (HCC) differ from healthy liver tissues and their heterogeneity may contain biological information associated with disease occurrence and clinical outcome that has yet to be fully evaluated at the proteomic level. This study provides a detailed description of the heterogeneity of NATs and the differences between NATs and healthy livers and revealed that molecular features of tumor subgroups in HCC were partially reflected in their respective NATs. Proteomic data classified HCC NATs into two subtypes (Subtypes 1 and 2), and Subtype 2 was associated with poor prognosis and high-risk recurrence. The pathway and immune features of these two subtypes were characterized. Proteomic differences between the two NAT subtypes and healthy liver tissues were further investigated using data-independent acquisition mass spectrometry, revealing the early molecular alterations associated with the progression from healthy livers to NATs. This study provides a high-quality resource for HCC researchers and clinicians and may significantly expand the knowledge of tumor NATs to eventually benefit clinical practice.

The maintenance of histone H3 lysine 9 trimethylation (H3K9me3) involves the recognition of preexisting modifications by heterochromatin protein 1 (HP1), which recruits the methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39H1) to methylate the adjacent newly incorporated histones, establishing a positive feedback loop. However, how this positive feedback is restricted to maintain H3K9me3 homeostasis remains largely unknown. We performed an unbiased genome-scale CRISPR-Cas9 screen and identified CUL5 ^ASB7 E3 ubiquitin ligase as a negative regulator of H3K9me3. ASB7 is recruited to heterochromatin by HP1 and promotes SUV39H1 degradation. During mitosis, cyclin-dependent kinase 1 (CDK1) phosphorylates ASB7, preventing its interaction with SUV39H1, leading to SUV39H1 stabilization and H3K9me3 restoration. Our findings reveal a dynamic circuit involving HP1, SUV39H1, and ASB7 that governs H3K9me3 homeostasis, ensuring faithful epigenetic inheritance and preventing excessive heterochromatin formation.