The Functions And Mechanisms Of M~6A Modification In Human Pancreatic Differentiation

Diabetes mellitus is a chronic metabolic disease,which shows a high incidence and severe growth trend all over the world.Diabetes is characterized by dysregulated glycemic conditions mainly due to the loss of functional pancreaticβcells.Human pluripotent stem cells(h PSCs)can differentiate into pancreatic cells and islet-like organoids which provides promising avenues for investigating pancreatic biology and treating diabetes.In order to further improve the differentiation efficiency andβcell function,we need to control the dynamic process more accurately.The in vitro pancreatic differentiation can simulates the development of human pancreas in vivo.As we all known,the pancratic development process is regulated by an intricate regulatory network containing transcription factors,signaling pathways and epigenetic modifications.N~6-methyladenosine(m~6A)is the most prevalent internal messenger RNA modification and plays pivotal roles in regulation of m RNA metabolism,while its functions in pancreatic differentiation and development remain elusive.Here,we profile the dynamic landscapes of m~6A modification transcriptome-wide during pancreatic differentiation for the first time,and find m~6A modification is highly dynamic,coordinating the global gene expression pattern.By analyzing the expression of key m~6A regulators,we focus our research on ALKBH5 the major m~6A demethylase.Firstly,we used the CRISPR-Cpf1 genome editing system to generate knockout h PSC lines of ALKBH5,and find that ALKBH5 plays significant regulatory roles in pancreatic differentiation and organogenesis.Mechanistic studies reveal that ALKBH5deficiency will lead to the increase of the m~6A modification on the m RNA of key pancreatic transcription factors(such as PDX1,NKX6.1,MNX1 and SOX9)and so on,thus promoting the binding of the recognition protein YTHDF2,which in turn mediates the degradation of m RNA.Interstingly,we further identify that ALKBH5 cofactorα-ketoglutarate(α-KG)can be applied to enhance differentiation by directly modulating the m~6A level.Collectively,our study profile the dynamic landscapes of m~6A modification transcriptome-wide during the differentiation of human pluripotent stem cells into pancreatic islet-like organoids for the first time,revealing their stage specificity and diversity;By constructing the ALKBH5 knockout human pluripotent stem cell lines and conducting in-depth research,we identify ALKBH5 as an essential regulator of pancreatic differentiation;This work reminder we can precisely control cell differentiation and developing by harnessing RNA methylation modification throughαKG-ALKBH5-m~6A-YTHDF2 axis.In conclusion,this study will help to establish a highly efficient differentiation system of pancreatic cells in vitro,thus laying the foundation for the development of new strategies for treating diabetes.