DNA Methylation–Dependent Restriction of Tyrosine Hydroxylase Contributes to Pancreatic β-Cell Heterogeneity

Abstract

The molecular and functional heterogeneity of pancreatic β-cells is well recognized, but the underlying mechanisms remain unclear. Pancreatic islets harbor a subset of β-cells that co-express Tyrosine Hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of the TH+ β-cells within islets is essential for appropriate function in mice, such that higher proportion of these cells corresponds to reduced insulin secretion. Here, we use these cells as a model to dissect the developmental control of β-cell heterogeneity. We define the specific molecular and metabolic characteristics of TH+ β-cells, and show differences in their developmental restriction in mice and humans. We show that TH expression in β-cells is restricted by DNA methylation during β-cell differentiation. Ablation of de novo DNA methyltransferase Dnmt3a in the embryonic progenitors results in a dramatic increase in the proportion of TH+ β-cells, while β-cell specific ablation of Dnmt3a does not. We demonstrate that maintenance of Th promoter methylation is essential for its continued restriction in postnatal β-cells. Loss of Th promoter methylation in response to chronic overnutrition increases the number of TH+ β-cells, corresponding to impaired β-cell function. These results reveal a regulatory role of DNA methylation in determining β-cell heterogeneity.

Differential CpG methylation atNnatin the early establishment of beta cell heterogeneity

“…Mice with global deletion of Nnat were used as previously described (67). Conditional Dnmt3a null mice (74), generated by crossing to Pdx1-Cre (75) have been described elsewhere (13, 15, 76). Mice containing the Dnmt3a floxed allele but lacking Pdx1-Cre were used as controls.…”

“…Sections were imaged using a TCS SP5 confocal microscope (Leica). Immunostaining of paraffin-embedded pancreas from mice with beta cell-selective deletion of Dnmt3a was performed according to (15, 33, 78) using antibodies against NNAT (ab27266, Abcam, 1:200) and INS (ab195956, Abcam, 1:800). Cell classification was carried out with a custom-written Fiji script (79) and CellProfiler pipeline (software version 4.1.3).…”

“…However, and overlaying this control, a second DMR at the Nnat promoter within the non-imprinted allele dictates beta cell subtype specificity of expression (Figure 2G). (via Pdx1-Cre) (15). In control mice, DNMT3A, but not DNMT1 expression, was detected in the majority of beta cells in late embryogenesis (E17.5) but was absent by postnatal day 7…”

“…Previous studies have provided us with an understanding of the transcriptional machinery that orchestrates beta cell development from early pancreatic and endocrine precursors (3)(4)(5)(6). To bolster these transcriptional programmes in vivo, chronic regulation via the epigenome appears to step in to maintain beta cell identity in the long term (7)(8)(9)(10)(11)(12)(13)(14)(15).…”

“…Identified subpopulations of beta cells have been associated with known markers or maturation states (Flattop/Cfap126 (26), PSA-NCAM (30), Cd81 (31), CD24 (32), TH (15), NPY (33)) are 'virgin' beta cells (34) or can be defined in terms of their roles in coordinating islet-wide Ca 2+ dynamics (e.g. 'hubs' (24) and 'leaders' (25,35,36)).…”