DNA methylation Dependent Restriction of Tyrosine Hydroxylase Contributes to Pancreatic -cell Heterogeneity

Abstract

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

Differential CpG methylation atNnatin the early establishment of beta cell heterogeneity

“…We describe here a novel subgroup of beta cells characterized by transient methylation of a second DMR (but not the gametic DMR) in the Nnat locus. This is consistent with previous findings that methylation at gametic DMRs is thought to be relatively stable [60,61], whereas 'secondary' imprinting regions have been shown to be more sensitive to nutrient-or physiology-based changes [10,62]. We have previously described the importance of Nnat for the normal secretory function of beta cells [45] and therefore hypothesise that NNAT + and NNATcells display differences in secretory function.…”

“…Dissected tissues were fixed, cryoprotected and embedded in optimal cutting temperature (OCT) and stored at -80 o C. Sections (10 µm) were cut using a CM1950 Cryostat (Leica) and immunostained using primary and secondary antibodies (listed in ESM Methods) and imaged using a TCS SP5 confocal microscope (Leica, Germany). Immunostaining of paraffinembedded pancreas from mice with beta cell-selective deletion of Dnmt3a was performed according to [10,28] as described in ESM methods. Human pancreata were processed, immunostained and imaged as described in ESM methods with the approval of the Ethics Committee of the University of Pisa, upon written consent of donors' next-of-kin.…”

“…To ask whether this apparent transition in the early postnatal period is driven by de novo CpG methylation, we assessed endogenous NNAT beta cell immunoreactivity in postnatal mice conditionally deleted for the methyltransferase DNMT3A at the pancreatic progenitor stage (using Pdx1-Cre) [10]. NNAT staining in control mice at postnatal (P) day 6 demonstrated expression in a subpopulation of beta cells whereas deletion of DNMT3A resulted in a loss of this heterogenous expression across the islet (Fig.…”

“…Previous studies have provided an understanding of the transcriptional machinery that orchestrates beta cell development from early pancreatic and endocrine precursors [3,4]. 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 [5][6][7][8][9][10][11].…”

“…Early reports [12][13][14], and more recent studies based on single cell transcriptomic profiling [15][16][17], electrophysiology [18] and functional imaging [19][20][21][22] have demonstrated functional heterogeneity amongst individual beta cells (reviewed in [23,24]). Identified subpopulations have been associated with known markers or maturation states (Flattop/Cfap126 [21], PSA-NCAM [25], CD81 [26], CD24 [11,27], TH [10], NPY [28], CD63 [29]), are 'virgin' beta cells [30] or are defined by their roles in coordinating islet-wide Ca 2+ dynamics (e.g. 'hubs' [19], 'leaders' [20] [31] and 'first responders' [32]).…”