The histone methyltransferase WHSC1 modulates keratinocyte proliferation in wounds via regulation of proliferation and differentiation marker expression

Summary: Abstract Body: Keratinocytes are structural cells in the skin that regulate wound repair through proliferation and differentiation, processes that are calcium (Ca)-dependent, with higher Ca levels promoting differentiation and inhibiting proliferation. In pathologic conditions, keratinocyte function is impaired, though the mechanisms remain unclear. Epigenetic chromatin modifying enzymes (CMEs) can control cell functions during tissue repair. Primary murine keratinocytes were cultured in low-Ca (LoCa) medium to isolate a basal subset, or differentiation was induced by switching to a high-Ca (HiCa) medium, and an unbiased epigenetic array was performed. HiCa-differentiated keratinocytes showed significantly decreased expression of WHSC1 (p<0.001), a histone methyltransferase that activates gene expression via K36me2 modification. Single-cell RNA sequencing of human wound keratinocytes revealed increased WHSC1 expression in cycling keratinocytes and decreased expression in differentiated keratinocytes, suggesting its role in proliferation. Further analysis of WHSC1-Hi versus WHSC1-Lo expressing cells revealed increased proliferation markers (e.g.,CDC7, E2F7, E2F2, PCNA) in WHSC1-Hi and increased differentiation markers (e.g.,VIM, FLG) in WHSC1-Lo. Further, siRNA and pharmacological inhibition of WHSC1, using siWhsc1 (1 μM) and LEM-14 (150 μM), respectively, in keratinocytes resulted in decreased E2f2, Cdc7 and increased Flg. To examine Ca-dependent regulation of WHSC1, keratinocytes were treated with Ca-sensing receptor inhibitor CalHex-231 (40 μM) and demonstrated increased Whsc1 and decreased Flg following HiCa-induced differentiation. Functionally, WHSC1 inhibition with LEM-14 reduced keratinocyte proliferation (measured via alamarBlue) (p<0.001) and decreased wound closure (p<0.0001) in scratch assays. Thus, WHSC1 plays a critical role in keratinocyte proliferation and may represent a potential therapeutic target for pathologic processes where keratinocyte function is impaired. Jadie Y. Moon¹, Amber L. Estor¹, Sonya J. Wolf¹, Amrita D. Joshi¹, James Shadiow¹, Tyler Bauer¹, Katherine A. Gallagher¹ 1. University of Michigan Michigan Medicine, Ann Arbor, MI, United States. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics