Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity
Authors: Yu Vanessa , Yong Fiona , Marta Angellica , Khadayate Sanjay , Osakwe Adrien , Bhattacharya Supriyo , Varghese Sneha S. , Chabosseau Pauline , Tabibi Sayed M. , Chen Keran , Georgiadou Eleni , Parveen Nazia , Suleiman Mara , Stamoulis Zoe , Marselli Lorella , De Luca Carmela , Tesi Marta , Ostinelli Giada , Delgadillo-Silva Luis , Wu Xiwei , Hatanaka Yuki , Montoya Alex , Elliott James , Patel Bhavik , Demchenko Nikita , Whilding Chad , Hajkova Petra , Shliaha Pavel , Kramer Holger , Ali Yusuf , Marchetti Piero , Sladek Robert , Dhawan Sangeeta , Withers Dominic J. , Rutter Guy A. , Millership Steven J. 
Published: March 2024

Abstract
Aims/hypothesis
Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected ‘hub’ cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion.

Methods
Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging.

Results
Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. ‘Hub’ cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation.

Conclusions/interpretation
These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.

Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

Graphical Abstract