Epigenetic regulation of the Meibomian gland in homeostasis and aging - SUMMARY The overall goal of this proposal is to delineate epigenetic mechanisms that control Meibomian gland homeostasis and are dysregulated in aging. The Meibomian gland’s key function is to secrete a lipid-rich film that coats the ocular surface and prevents tear film evaporation. Aged Meibomian glands show reduced size and decreased proliferation of basal progenitor cells. These phenotypes are thought to contribute to Meibomian gland dysfunction in aging, including development of Evaporative Dry Eye Disease (EDED) which is common in aged patients, can seriously impair vision and currently has no effective treatment. Histone deacetylases (HDACs) are druggable targets with therapeutic potential and play key roles in controlling progenitor cell activity and maintenance in other epithelia. HDACs function by modifying chromatin and by controlling the activities of key transcription factors. Our published data show that inducible Hdac1 and Hdac2 deletion in adult Meibomian gland basal cells causes decreased proliferation and Meibomian gland loss. The transcription factor GLI2, a key component of the Hedgehog pathway, is hyper-acetylated in Hdac1/2 mutant Meibomian glands, a modification that is known to reduce GLI2’s transcriptional activity and association with active chromatin. Deletion of the Hedgehog co-receptor Smo causes decreased basal proliferation and Meibomian gland hypoplasia, partially mimicking Hdac1/2 loss, while epithelial deletion of the Hedgehog inhibitory co-receptor Ptch1 or forced expression of activated GLI2 in Meibomian gland basal cells results in acinar basal cell expansion, identifying Hedgehog signaling and GLI2 as key regulators of Meibomian gland proliferation. Comparative analysis of single nuclear RNA-seq data from the eyelid tarsal plates of young and old mice showed that Hedgehog signaling is decreased in aged compared with young Meibomian glands. This decline is associated with increased acetylation of GLI2 and histone H3, suggesting that HDAC1/2 activity is decreased in aged Meibomian glands. By contrast, Hdac3 deletion increases basal progenitor cell proliferation and GLI2 expression but not its acetylation levels, like the effects of elevated Hedgehog signaling. Based on these data we hypothesize that HDAC1/2 and HDAC3 play distinct and key roles in adult Meibomian gland progenitor cells in part by differentially modifying the expression and activity of Hedgehog pathway components and target genes, and that decreased HDAC1/2 activity contributes to aging phenotypes. To test this hypothesis and delineate additional mechanisms in an unbiased fashion, we will use genetic and single- cell genomics approaches to identify the direct targets and transcriptional co-regulators of HDAC1/2 and HDAC3 in Meibomian gland progenitor cells and determine the extent of overlap in HDAC1/2-regulated genes with those dysregulated in aging. These experiments have potential to reveal novel therapeutic strategies to enhance progenitor cell activity in aged Meibomian glands.
