Geminin (Gmnn) is a coiled-coil nuclear protein originally identified as a DNA replication inhibitor. In addition, nuclear Gmnn regulates gene expression by interacting (through a domain distinct from the Cdt1-interacting domain) with Brg1, the catalytic subunit of the SWI/SNF chromatin remodeling complex. In this context, Gmnn regulates expression of key transcription factors involved in differentiation. The dual role of Gmnn in the cell cycle and differentiation has been viewed as a critical mechanism for linking replication and differentiation. We wanted to test its role in human pancreas development. We found a robust Gmnn expression in the human fetal pancreas but its expression slightly decreased with age. Remarkably the fraction of cells expressing both Gmnn and Ki67 declined much faster with age. We isolated 13WD human fetal pancreas and studied the proportion of replicating Sox9+cells. We compared control human fetal explants with fetal explants transduced with a shRNA lentivirus directed against Gmnn (Gmnnsh) and discovered that Gmnnshexplants display a decrease in proliferation compared to controls after 2 days in culture. RNA sequencing comparing Gmnnshexplants to controls revealed a marked decrease in transcripts associated with endocrine differentiation. To evaluate the effects on endocrine differentiation, human fetal pancreases were isolated at 13WD and treated for 5 days with lentiviruses or casein kinase inhibitors. We detected a significant reduction in the number of insulin+cells after treatment with the casein kinase inhibitor TBB and a significant reduction in the number of glucagon+cells after treatment with TBB or after siCsnk2a1. Overexpression of Gmnn significantly increased the proportion of Neurog3+cells compared to the control explants.
Finally, we found that inhibition of Gmnn or casein kinase 2 was capable of significantly reducing expression of Nkx6.1 compared to controls. We conclude that Gmnn may play a critical role pancreas cell replication and endocrine specification in human development.
L. Baeyens: None. D.W. Scheel: None. K. Yang: None. M. German: Advisory Panel; Self; Encellin. Stock/Shareholder; Self; Encellin, Viacyte, Inc.