SYSTEMATIC REVIEW published: 28 August 2020 doi: 10.3389/fcell.2020.00748

Roles of Epigenetic Modifications in the Differentiation and Function of Pancreatic β-Cells

Fei Xu 1,2, Jing Liu 3, Lixin Na 2,3 and Linjun Chen 3*

1 Department of Microbiology and Immunology, Shanghai University of Medicine & Health Sciences, Shanghai, China, 2 Collaborative Innovation Center of Shanghai University of Medicine & Health Sciences, Shanghai, China, 3 Department of Inspection and Quarantine, Shanghai University of Medicine & Health Sciences, Shanghai, China

Diabetes, a metabolic disease with multiple causes characterized by high blood sugar, has become a public health problem. Hyperglycaemia is caused by deficiencies in insulin secretion, impairment of insulin function, or both. The insulin secreted by pancreatic β cells is the only hormone in the body that lowers blood glucose levels and plays vital roles in maintaining glucose homeostasis. Therefore, investigation of the molecular mechanisms of pancreatic β cell differentiation and function is necessary to elucidate the processes involved in the onset of diabetes. Although numerous studies have shown that transcriptional regulation is essential for the differentiation and function of pancreatic β Edited by: cells, increasing evidence indicates that epigenetic mechanisms participate in controlling Daniel Enquobahrie, University of Washington, the fate and regulation of these cells. Epigenetics involves heritable alterations in United States gene expression caused by DNA methylation, histone modification and non-coding Reviewed by: RNA activity that does not result in DNA nucleotide sequence alterations. Recent Hong Zan, research has revealed that a variety of epigenetic modifications play an important role The University of Texas Health Science Center at San Antonio, United States in the development of diabetes. Here, we review the mechanisms by which epigenetic Pandora L. Wander, regulation affects β cell differentiation and function. University of Washington, United States Keywords: epigenetics, pancreatic β cells, histone modification, DNA methylation, non-coding RNA *Correspondence: Linjun Chen [email protected] INTRODUCTION

Specialty section: The global incidence of diabetes has risen sharply with societal progress and improvements This article was submitted to in living standards, and the disease is showing a trend toward affecting younger individuals. Epigenomics and Epigenetics, At present, ∼1/10th of the world’s medical expenses are used for the treatment of diabetes a section of the journal and its complications, imposing heavy economic burdens on patients’ families and on society Frontiers in Cell and Developmental as a whole (Hassan et al., 2017). Diabetes and its complications are common and challenging Biology global problems in the twenty-first century. Diabetes, which is a chronic metabolic disease Received: 03 June 2020 characterized by elevated blood sugar, is caused by the interaction of multiple factors, including Accepted: 17 July 2020 genetic, environmental, social, and psychological factors, and among others (Kolb and Martin, Published: 28 August 2020 2017). Persistent hyperglycaemia causes chronic damage to various organs, including the eyes, Citation: kidneys, heart, blood vessels, and nerves, which can eventually lead to patient death due to organ Xu F, Liu J, Na L and Chen L (2020) failure (Zimmet et al., 2016). According to the pathogenesis, diabetes is mainly divided into two Roles of Epigenetic Modifications in the Differentiation and Function of types: Type 1 diabetes (T1D) is caused by an autoimmune attack targeting the insulinproducing Pancreatic β-Cells. pancreatic b-cells, while type 2 diabetes (T2D) is associated with aging, early development of Front. Cell Dev. Biol. 8:748. insulin resistance, and a deteriorating β cell function. T2DM is a complex disease resulting from doi: 10.3389/fcell.2020.00748 the interaction of genetic, epigenetic, environmental, and lifestyle factors. About 90% of diabetic

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cases worldwide are accounted to be T2D and predominantly TABLE 1 | Members and functions of DNA methylated transferases. targets adults. Furthermore, T2DM can be complicated with Name Location Functions chronic conditions, such as hypertension, coronary heart disease, stroke, macrovascular and microvascular complications, DNMT1 19p13.2 The main enzyme to maintain methylation which which has greatly challenged the global medical and health ensures the normal replication of DNA methylation system; thus, the prevention and treatment of diabetes and its mode; and also necessary for de methylation of non complications have become popular research topics worldwide CpG sites (Tajima et al., 2016) DNMT2 10p15.1 The methyltransferase of aspartic tRNA, which can (Ueda et al., 2019). ◦ Epigenetics was originally proposed by Waddington in 1942, methylate 38 C of aspartic tRNA (Goll et al., 2006) and its definition was finalized at the 2008 Cold Spring DNMT3A 2p23.3 The main de novo DNA methyltransferase which establishes DNA methylation patterns in gametes Harbor Conference as “a stable heritable phenotype caused by and early embryos (Chedin, 2011; de Mendoza chromosomal changes without changing the DNA sequence” et al., 2018) (Berger et al., 2009; Waddington, 2012). Researches have revealed DNMT3B 20q11.2 The main de novo DNA methyltransferase which that the mechanisms of these changes mainly comprise DNA establishes DNA methylation patterns in gametes methylation, histone modification and noncoding RNA (ncRNA) and early embryos (Chedin, 2011) activity (Ling and Ronn, 2019). Epigenetic regulation exists DNMT3L 21q22.3 Has no methyltransferase activity by itself, interacts throughout the lifetime of an organism and participates in with the DNMT3A and DNMT3B catalytic regions to enhance the activity of DNMT3A and DNMT3B, thus many life activities. Any abnormality in the epigenetic regulatory facilitating de novo methylation (Chedin, 2011) mechanism will affect chromatin structure and gene expression, leading to the occurrence of a variety of diseases (Nilsson et al., DNMT1, DNA methyltransferase 1; DNMT2, DNA methyltransferase 2; DNMT3A, DNA methyltransferases 3A; DNMT3B, DNA methyltransferases 3B; DNMT3L, DNA 2018; Li S. et al., 2019; Perez and Lehner, 2019). Epigenetic methyltransferases 3L. changes are important regulatory mechanisms of islet β cell function and play irreplaceable roles in the normal physiological function of pancreatic β cells and insulin-secreting cells and apoptosis (Courty et al., 2019; Gong and Jiang, 2020; Makkar elements in the genome, which in turn affect the development et al., 2020). There is no doubt that epigenetics research already of the disease (Jirtle and Skinner, 2007). Research has shown becomes the focus of the study of pancreatic β cells in recent that glucose metabolism disorders in T2DM patients are related years. Epigenetic modifications are closely related to other to the hypomethylation of DNA in peripheral blood leukocytes biological processes in the physiological function of pancreatic (Toperoff et al., 2015). Another example is in adults exposed to β cells. If the epigenetic changes are moderately induced or famine during the Dutch Hunger Winter in the late period of controlled by drug intervention, it is expected to become a new World War II. The offspring of these individuals presented a low- point to prevent the occurrence and development of diabetes. birth weight, as well as an increase in the incidence of obesity, T2DM and dyslipidemia (Barres and Zierath, 2016). The DNA methylation level of genes related to cardiovascular disease in EFFECTS OF DNA METHYLATION ON β peripheral blood leukocytes of diabetic patients was significantly CELL DIFFERENTIATION AND FUNCTION higher than that of normal people, and the DNA methylation level in saliva of diabetic nephropathy patients was significantly DNA methylation modification is by far the most thoroughly higher than that of ordinary people (Babu et al., 2015; Ronn studied epigenetic modification (Shafabakhsh et al., 2019). and Ling, 2015). In addition, studies have shown that the long- DNA methylation modification is mainly controlled by DNA term exposure of insulin and glucose will seriously change the methyltransferase (DNMT) family proteins (Table 1) (Sahin DNA methylation status of skeletal muscle, indicating that DNA et al., 2010). S-adenosylmethionine is used as the methyl donor methylation is a rapid adaptation epigenetic marker (Mudry to methylate the cytosine on CpG islands. Normally, the CpG et al., 2017). island of a gene is in an unmethylated state. Methylation of the The methylation of the CpG island in the insulin promoter cytosines in the CpG island can inhibit the expression of this region may play a crucial role in the maturation and tissue- gene (Figure 1). Increasing evidence shows that the regulation specific expression of insulin genes in pancreatic β cells. of methylation level is related to diet, physiological activities and Research scholars at Malmö University Hospital in Sweden glucose level, indicating that methylation is a dynamic process found that in the islets of T2DM patients and those of non- (Wicklow and Sellers, 2015). There is evidence that both the T2DM donors, insulin promoter DNA methylation in human density and methylation level of tissue-specific promoter CpG islets was negatively correlated with insulin gene expression and islands play important roles in the regulation of gene expression positively correlated with HbA1c (Yang et al., 2011). Relatedly, (Bansal and Pinney, 2017; Ponnaluri et al., 2017; Zhou et al., insulin methylation levels increased and expression decreased 2018). in T2DM patients (Yang et al., 2011). Dayeh et al. performed DNA methylation chip array analysis on islets donated by 15 DNA Methylation in Diabetic Patients T2DM patients and 34 normal controls and found that 853 Both the environment and diet can lead to apparent changes genes and 1,649 CpG sites were differentially methylated in in the modification of certain imprinted genes and transposable the islets of diabetic patients (Dayeh et al., 2014). In vitro

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FIGURE 1 | Molecular mechanism of DNA methylation (Bansal and Pinney, 2017; Ponnaluri et al., 2017; Zhou et al., 2018; Shafabakhsh et al., 2019). DNA methylation modification is mainly controlled by DNMT proteins. S-adenosylmethionine is used as the methyl donor to methylate the cytosine on CpG islands. Normally, the CpG island of a gene is in an unmethylated state. Methylation of the cytosines in the CpG island can inhibit the expression of this gene.

experiments confirmed that methylation of the CDKN1A and causing abnormal Arx expression in β cells (Dhawan et al., 2011). PDE7B promoter genes inhibited their transcriptional activity In β cells, the inhibition of this methylation mediator ArxTat and led to a decrease in the exocytosis function of pancreatic β occurs through the interaction of NK2 homeobox 2 (Nkx2.2) cells and reduced insulin secretion (Dayeh et al., 2014). These with an inhibitory complex containing Grg3 (also known as epigenetic changes provide further insight into the pathogenesis Tle3), HDAC1 and Dnmt3A (Papizan et al., 2011). Therefore, of diabetes. in current cell replacement therapy research, endogenous alpha cells are considered to be important for β cell reprogramming. DNA Methylation in β Cell Differentiation Recent studies have also shown that inhibiting DNA methylation and Function in pancreatic progenitor cells promotes alpha cell production The role of DNA methylation in early pancreatic development (Liu et al., 2019). In addition, the hypermethylation of CpG is unclear, and Anderson et al. found in a model of induced islands can reduce the expression of HNF4α gene and affect the pancreatic β cell ablation that DNA methylation in pancreatic differentiation of pancreatic β cells (Gilbert and Liu, 2012). progenitor cells plays an important role in the differentiation PDX1 mutations can cause special types of diabetes, showing of pancreatic progenitor cells (Anderson et al., 2009). The that PDX1 silencing can promote pancreatic β cell damage specific knockout of DNMT1 in mouse pancreatic progenitor leading to diabetes (Pedica et al., 2014). The DNA methylation cells showed pancreatic progenitor cell apoptosis and pancreatic of 10 CpG sites in the PDX1 promoter and enhancer regions of hypoplasia (Georgia et al., 2013). In mature β cells, defects in pancreatic islets in T2DM patients was increased compared with DNMT1 or DNMT3 can cause β cells to lose their “identity” and the control group. Pancreatic β cells exposed to hyperglycaemia be reprogrammed into alpha cells, indicating that inhibition of showed increased DNA methylation and decreased expression of alpha cell programming is necessary to maintain the “identity” PDX1 in the islets. Overall, the epigenetic modification of PDX1 of β cells (Dhawan et al., 2011). Currently, research has found may play a role in the development of T2DM. that the occurrence of this reprogramming is related to the Peroxisome proliferator-activated receptor γ coactivation 1α methylation of the Aristaless-related homeobox (Arx) promoter, (PPARGG1α) is a transcriptional coactivator with high levels of

Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 August 2020 | Volume 8 | Article 748 Xu et al. The Epigenetic Regulation in Pancreatic β Cell expression in the human liver, kidneys, pancreas and skeletal expressed in the murine adult pancreas, yet the pancreas muscles. DNA methylation of the PPARGG1α promoter may be has lower genomic 5hmC levels than other adult tissues an important cause of diabetic cardiopathy (Lacquemant et al., derived from endoderm, including liver and lung (Ito et al., 2000; Waldman et al., 2018). Ling et al. reported that the DNA 2010, 2011), suggesting dynamic DNA demethylation during methylation of the PPARGG1α promoter in the islets of T2DM pancreas development. Xianghui Fu et al. found that TETs patients was accompanied by decreased mRNA expression, and TDG are direct targets of miR-26a and the expression suggesting that epigenetics can regulate the expression of the of TETs and miR-26a change in opposite directions during PPARGG1α gene and subsequently affect insulin secretion (Ling in vivo and in vitro pancreatic cell differentiation (Fu et al., et al., 2008). 2013). Another study showed that IFN-α exposure causes DNA TCF7L2 is a T2DM susceptibility gene that can promote the demethylation by upregulation of the exoribonuclease PNPase proliferation and survival of pancreatic β cells, and regulate the old-35 (PNPT1), which mediates degradation of miR-26a leading function of glucagon-like peptide (GLP-1) synthesis by intestinal to overexpression of methylcytosine dioxygenase TET2 and L cells. Hu et al. performed high-throughput detection on increased 5-hydroxymethylcytosine levels in human islets and β pancreatic islet cells cultured in high fat and high glucose and cells. These results provide a mechanistic framework to explain found that chronic glycolipid toxicity can induce abnormal DNA how inflammatory triggers such as viral infections can modify methylation of the TCF7L2 gene, which may be one of the the β cell epigenome and modulate the interactions between mechanisms of glycolipid toxicity leading to the deterioration of the immune system and β cells (Stefan-Lifshitz et al., 2019). diabetic islet cells (Hu et al., 2014). Therefore, the role of dynamic DNA demethylation during In addition, DNA methylation is considered to be an pancreas development deserves further study. important intergenerational genetic mechanism (Sarkies, 2020). DNA methylation markers in paternal and maternal genomes THE ROLE OF HISTONE MODIFICATION IN undergo reprogramming during mammalian fertilization and embryonic development; however, some imprinted genes escape β CELL DIFFERENTIATION AND FUNCTION the demethylation process during this time and thus change the The core histones in the chromatin structure are subject representative type, resulting in intergenerational inheritance. to a series of covalent modifications with different chemical In 2015, a study of perinatal female rat (F0) exposured to groups (including acetylation and methylation) that lead to bisphenol A showed that the methylation of the imprinted the formation of a modified cascade called a histone code. gene insulin-like growth factor 2 (Igf2) of the F2 generation The histone code can be recognized by a series of protein can be changed and cause intergenerational inheritance to complexes, is translated into a specific chromatin state, and has impaire insulin secretion and glucose intolerance of the a very important gene transcription regulatory function (Sims offspring (Mao et al., 2015). Su Rina et al. established a and Reinberg, 2008). The ease of access to the transcription rat model of moderate intrauterine hyperglycemia induced factor binding site on chromatin is determined by the by streptozotocin to detect glucose and lipid metabolism of position and encapsulation of histones, and the degree of first-generation (F1) and second-generation (F2) offspring. nucleosome compression is determined by the activity of a The results showed that F1 and F2 offspring which were series of enzymes that are responsible for various histone-specific exposed to intrauterine hyperglycemia had impaired insulin covalent modifications (such as acetylation, methylation, and secretion from the islets, and both F1 and F2 offspring showed phosphorylation) (Berger, 2007). The most studied types of similar hypomethylation level at the−1952 site of the tumor histone modifications are acetylation and methylation of specific necrosis factor (Tnf) gene (Su et al., 2016). These results amino acid residues of histones H3 and H4 (Figure 2). Related confirmed that DNA methylation occurs in offspring exposed research shows that during the differentiation and development to intrauterine hyperglycemia and that the DNA methylation is of β cells, histone modification plays an important regulatory role intergenerational and inherited. Ten eleven translocation (TET) enzymes and thymine DNA and can recruit protein complexes to participate in the regulation β glycosylase (TDG) are implicated in active DNA demethylation of the expression of related genes in cells (Ahmed et al., 2017; (Ito et al., 2010). The three TET family enzymes oxidize 5- Balaji et al., 2018; Makkar et al., 2020). methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), and subsequently to 5-formylcytosine (5fC) and 5- Histone Methylation carboxylcytosine (5caC) (Ito et al., 2010; He et al., 2011). Histone methylation often occurs at the lysine or arginine TDG, a base excision repair glycosylase, replaces 5fC and residues of histone H3 and H4. Such methylation is cocatalysed 5caC with an unmodified cytosine via DNA repair (He by histone methyltransferases (HMTs) and histone demethylase et al., 2011). Although recent data suggest a role of TET 1 (lysine demethylase 1, LSD1), indicating that the histone and 5hmC in embryonic stem cells and primordial germ methylation process is reversible (Jenuwein, 2006). Histone cells, evidence for enzymatic demethylation by TET enzymes methylation is more stable and persistent than histone during differentiation of cells of later stages, such as the acetylation. Histone methylation of different forms and at postnatal and adult stem cells of various organs including different amino acid sites can lead to activation or inhibition of pancreas, remains very limited (Auclair and Weber, 2012; gene transcription (Marmorstein and Trievel, 2009). The forms Tan and Shi, 2012). Interestingly, TET2 and TET3 are highly of histone methylation include monomethylation, dimethylation,

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FIGURE 2 | Molecular mechanism of histone modification (Ahmed et al., 2017; Balaji et al., 2018; Makkar et al., 2020). (A) The histone acetylation process consists of HATs and HDACs. HATs can catalyse histone acetylation, resulting in the relaxation of chromatin structure, and makes it easy to recruit transcription factors to combine with them and promote the transcriptional expression of genes. On the contrary, HDACs deacetylate histones to lead to chromatin compression and inhibit gene transcription. HATs and HDACs jointly participate in the dynamic equilibrium processes of histone acetylation to precisely regulate gene transcription, such as for histone H3 at positions 9, 14, 18, and 23. Once the dynamic equilibrium state of acetylation and deacetylation of histones is broken, it may cause disease. (B) Histone methylation often occurs at the lysine or arginine residues of histone H3 and H4. Histone methylation is more stable and persistent than histone acetylation. Histone methylation of different forms and at different amino acid sites can lead to activation or inhibition of gene transcription. The forms of histone methylation include monomethylation, dimethylation, and trimethylation. Histone lysine methylation is relatively stable and often occurs at positions H3K4, H3K9, H3K27, H3K36, and H3K79. and trimethylation. Histone lysine methylation is relatively stable Histone Deacetylase Inhibitors (HDACis) and often occurs at positions H3K4, HK9, H4K20, H4K27, and Pancreatic β Cells H3K36, and H3K79 (Table 2). HDACis can improve the function of pancreatic β cells, promote insulin synthesis and release, reduce inflammatory response- Histone Acetylation induced damage to pancreatic β cells, regulate the influence The histone acetylation process consists of histone of the systemic immune response on T1DM and T2DM, and acetyltransferases (HATs) and histone deacetylases (HDACs) ameliorate the complications of diabetes (Sharma and Taliyan, (Ahmed et al., 2017). It is known that HATs can catalyse histone 2016a). Short-chain fatty acids (such as butyric acid) that act as acetylation, resulting in the relaxation of chromatin structure, HDACis are intestinal fermentation products that exist in natural and makes it easy to recruit transcription factors to combine foods and may have broad application prospects in the treatment with them and promote the transcriptional expression of of GDM. genes. On the contrary, HDACs deacetylate histones to lead to HDACis can be divided into 6 categories, according to chromatin compression and inhibit gene transcription. HATs their chemical structure (Table 3): ① short-chain fatty acids, and HDACs jointly participate in the dynamic equilibrium such as sodium valproate (VPA) and sodium butyrate (NaB); processes of histone acetylation to precisely regulate gene ② hydroxamic acids, such as trichostatin A (TSA) and transcription, such as for histone H3 at positions 9, 14, 18, suberoylanilide hydroxamic acid (SAHA); ③ cyclic tetrapeptides, and 23 (Gilbert and Liu, 2012). Once the dynamic equilibrium such as trapoxin A, (TPX); ④ benzamides, such as entinostat state of acetylation and deacetylation of histones is broken, it (MS-275); ⑤ epoxy ketones, such as trapoxin B; and ⑥ hybrid may cause disease. It was previously shown that the level of molecules, such as peptide hydroxamic acids (cyclic hydroxamic H3K9 acetylation in the promoter region of insulin receptor acid-containing peptide 31, CHAP31) (Christensen et al., 2011; substrate 2 (IRS2) was positively correlated with the expression Sharma and Taliyan, 2016a). Mosley et al. verified the effect level of IRS2 in pancreatic β cells (Kawada et al., 2017). High of glucose concentration on the acetylation of the insulin gene expression of HDAC7 impairs the secretory ability of rat promoter in the islet β cell line MIN6. Their study revealed islet cells, and the expression level of islet HDAC7 in T2DM that the histone acetylation level of the glucose promoter was patients is significantly increased compared with healthy group significantly greater when the glucose concentration was 30 (Daneshpajooh et al., 2017). mmol/L than when it was 3 mmol/L. After treatment with the

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TABLE 2 | Histone-lysine methyltransferase in the differentiation and function of and NF-κB inhibitor genes (Larsen et al., 2007; Lundh et al., pancreatic β cells. 2010). In animal models of diabetes, the inflammatory response

Genes Action sites Targets Functions can inhibit the survival of pancreatic β cells, and many HDACis (e.g., TSA and SAHA) can reduce cytokine-induced inhibition EHMT2 or G9a H3K9me3, HMGA1 Gene silencing (Cao et al., 2019) of insulin secretion. Inhibiting the inflammatory response is H3K27 considered a key step in patients undergoing pancreatic β cell SETDB1 H3K9me3 PPARγ, Gene silencing (Okamura et al., transplantation for diabetes (Lundh et al., 2010). CEBPα 2010) SET7/9 H3K4 HIF-1α, Gene silencing (Baek and Kim, 2016) Histone Modification and Pancreatic β Cell p21, PDX1, Gene activation (Maganti et al., Development and Function BETA2 2015) Studies have shown that the modification of histones determines SET8 H4K20 / Block ROS accumulation, attenuat vascular inflammation, and restore whether pluripotent pancreatic progenitor cells produce β cells. nitric oxide production (Yao et al., Haumaitre et al. studied the regulatory role of HDAC expression 2014) and activity in the development of rat pancreases and found that PRC2 H3K27 Insulin gene Inhibit the expression of insulin HDAC inhibitors can reduce the differentiation of pancreatic (Pethe et al., 2014) exocrine cells and increase the number of ductal cells and Ngn3- RAD21 Increased MafA expression (Deering positive endocrine progenitor cells (Haumaitre et al., 2009). The et al., 2009) dynamic changes in histone acetylation on the final different PDX1 Inhibit the differentiation of β cells types of endocrine cells (that is, the number of α-, β-, δ-, ε-, (Chang et al., 2016) and PP cells) have a decisive role. The different types of HDAC LncRNA Increased VEGF expression ANRIL (Thomas et al., 2017) have different effects on the development of pancreatic cells. miR-200b Increased VEGF expression (Ruiz For example, class I HDAC members specifically inhibit the et al., 2015) production of α- and PP cells while promoting the differentiation SUV39H1 H3K9 p53 Gene silencing (He et al., 2012) of β- and δ-cells (Haumaitre et al., 2008). The genetic deletion KMT2D/MLL2 H3K4 / Regulate glucose homeostasis of different members of class II HDAC can lead to an increase (Scoville et al., 2015) in β cells and/or δ-cells, and their overexpression can cause the KMT2C/MLL3 numbers of β- and δ-cells to be reduced (Lenoir et al., 2011). KMT2B/MLL4 Therefore, histone modification enzymes have a highly specific NSD2 H3K36 PDX1 Promote the differentiation of β cells role during the development and differentiation of different (Poulin et al., 2016) pancreatic cells. In addition, studies have shown that the HDAC EZH2 H3K27me3 INK4a/ARF Gene silencing (Chen et al., 2009) inhibitor MC1568 can promote the expression of PDX4 and insulin (Dayeh and Ling, 2015). These results show that HDAC HMGA1, high mobility group at-hook 1; BDNF, brain derived neuro trophic factor; PPARγ, peroxisome-proliferator activated receptor γ; CEBPα, CCAAT-enhancer binding protein α; plays a key role in mediating pancreatic β cell differentiation HIF-1α, hypoxia inducible factor-1α; PDX1, pancreatic and duodenal homeobox 1; BETA2, and development. beta cell E-box transcription factor 2; SUV39H1, suppressor of variegation 3-9 homolog A series of transcription factors, such as PDX1, require 1; KMT2D, lysine methyltransferase 2D; KMT2B, lysine methyltransferase 2B; KMT2C, chromatin modification factors to effectively regulate gene lysine methyltransferase 2C; NSD2, nuclear receptor binding SET domain protein 2; EZH2, enhancer of zestehomolog 2; INK4a/ARF, inhibitor of CDK4/ alternative reading frame. transcription, which is essential for maintaining the normal function of β cells. If histone modification changes, it may cause a series of genes in β cells to exhibit high or low expression. This may further cause the occurrence of diabetes. HDACi TSA, the degree of H3 and H4 acetylation on the insulin Chakrabarti et al. found that β cells are highly methylated gene promoter in the 3 mmol/L glucose group was similar to and acetylated at the H3K4 proximal promoter region of that in the 30 mmol/L glucose group, and the mRNA expression the insulin gene, and this modification is highly correlated of insulin in the cells cultured with glucose was similar. These with the recruitment of the histone methyltransferase Set7/9 results show that TSA and NaB can promote insulin promoter and the histone acetyltransferase P300, which suggests that histone acetylation to accelerate insulin transcription (Mosley the expression of the insulin gene is regulated by histone et al., 2004). In addition, studies have found that IL-1, TNF- modification (Chakrabarti et al., 2003). PDX1 can recruit the α and other inflammatory cytokines are closely related to islet histone methyltransferase Set7/9, which in turn methylates H3K4 cell damage and can affect the expression of multiple genes in in the promoter region of the insulin gene, to promot the pancreatic β cells, including cell apoptosis, cell cycle regulation, expression of the insulin gene (Maganti et al., 2015). Another cell differentiation and metabolism (Ortis et al., 2010). After study showed that Set7/9 is necessary for normal β cell function. HDACi intervention, IL-1β- or TNF-α-induced apoptosis of Set7/9 can regulate insulin secretion-related genes (such as cultured β cells in vitro is inhibited, which protects pancreatic Ins1/2, Glut2, and MafA) with transcription factor PDX1 and β cells from cytokine toxicity. HDACis bind to oligonucleotides RNA polymerase II (Deering et al., 2009). Related studies also to form complexes that reduce the transcription of NF-κB- show that under high glucose stimulation, p300 and PDX1 dependent genes, such as inducible nitric oxide synthase (iNOS) cooperate to promote insulin transcription of β cells; on the

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TABLE 3 | Effects of HDACis in preclinical animal models of insulin resistance.

HDAC inhibitor Dosage regimen Animal model Potential benefit

NaB 5% w/w for 16 weeks HFD-fed mice Increased insulin sensitivity; Increased energy expenditure (Gao et al., 2009) 500 mg/kg, i.p. HFD-fed mice Increased energy expenditure (Li et al., 2012) 5% w/w for 4 weeks C57BL/6N mice Improved insulin sensitivity (Lin et al., 2012) 400 mg/kg/day for 10 weeks; i.p HFD + Low dose Reduced insulin resistance (Khan and Jena, 2016a) TSA 0.6 µg/kg/day for 12 weeks HFD-fed mice Reduced adiposity (Li et al., 2012) 0.8 mg/kg, i.p. HFD-fed mice Induction of Fibroblast growth factor-21 (Li et al., 2012) MS-275 10 mg/kg i.p. C57BLKS/ J-Leprdb/db Increased Energy expenditure; Increased insulin sensitivity (Galmozzi et al., 2013) SAHA 25 mg/kg i.p. every other day for 23 days C57BLKS/ J-Leprdb/db Increased Energy expenditure; Increased insulin sensitivity (Galmozzi et al., 2013) VPA 150–300 mg/kg/day for 3 weeks; orally STZ (75 mg/kg, i.p.) in SD rats Increased β-cell proliferation (Khan and Jena, 2016b) 150–300 mg/kg/day for 10 weeks; orally HFD + Low dose STZ Reduced β cell damage; Reduced insulin resistance (Khan et al., 2016) SAHA 25–50 mg/kg i.p. Swiss albino mice fed with HFD for Improved insulin resistance (Sharma and Taliyan, 2016b) 8 weeks ITF 2357 1.25–2.5 mg/kg; orally Female C57BL/6 mice Reduced β cell toxicity; Decreases Serum Nitrite (Lewis et al., 2011)

contrary, at low glucose levels, HDAC1 and HDAC2 bind in repression of the Ink4a/Arf locus and augmented β- to PDX1 to inhibit insulin transcription (Mosley and Ozcan, cell proliferation. 2004; Wang et al., 2007). In addition, the Swi/Snf chromatin The PcG protein Ezh2, functioning as a HMT in remodeling complex can be involved in the regulation of blood PolycombRepressive complex 2 (PRC2), was shown to regulate glucose by PDX1. Brg1-Swi/Snf can be used as a co-activator of islet cell growth by influencing the proliferative potential of PDX1-mediated gene expression in β cells, while Brm-Swi/Snf β cells (Chen et al., 2009). Conditional deletion of Ezh2 in β can be used as a co-suppression method. The antagonistic cells led to decreased H3K27me3 at the Ink4a/Arf locus. This effects of Brg1-Swi/Snf and Brm-Swi/Snf affect the expression induces a replication failure of β cells in neonatal mice, a reduced of target genes in β cells, which is important for maintaining β- cell mass and mild diabetes. Ezh2, by regulating histone glucose homeostasis. Chang et al. also found that PDX1 histone methylation and repression of the Ink4a/Arf locus in pancreatic modification of the promoter region H3K4, such as deacetylation, islets, permit physiological β-cell expansion in neonatal mice, demethylation and other histone modifications, can lead to and adaptive β-cell regeneration after conditional chemical downregulation of PDX1 in pancreatic β cells (Chang et al., ablation of β cell in adults. This shows the importance of 2016). Studies have also shown that MLL3 and MLL4 bind to the epigenetic mechanisms in the regulation of β-cell proliferation, transcription factors MafA and MafB to regulate pancreatic β cell and suggests that epigenetic manipulation of PcG proteins and function (Scoville et al., 2015). their respective histone modification marks in β cells might be a The products of the Ink4a/Arf (Cdkn2a) locus, the cyclin- useful tool for promoting β-cell proliferation. Altogether, these dependent kinase inhibitor p16INK4a and the tumor suppressor studies provide evidence that histone modifications, regulated by p19Arf showed increased expression with age and have been complexes containing TrxG and PcG proteins, are required for linked to reduction in the proliferative capacity of aged β cells specific gene repression and permit physiological and adaptive (Krishnamurthy et al., 2006). The locus Cdkn2a has indeed β-cell expansion. been identified linked to T2DM by genome-wide association analysis (Diabetes Genetics Initiative of Broad Institute of et al., 2007). Interestingly, a recent study reported that Bmi- THE ROLE OF NONCODING RNA IN β 1 dependent modulation of the Ink4a/Arf expression levels CELL DIFFERENTIATION AND FUNCTION is critical to regulate pancreatic β-cell proliferation during aging and regeneration (Dhawan et al., 2009). Loss of Bmi- The Role of miRNAs in β Cell 1 resulted in reduced β-cell proliferation. Decreased Bmi- 1 Differentiation and Function binding to the Ink4a/Arf locus in aged islet led to reduced MicroRNA is a type of endogenous noncoding small, single- H2A ubiquitination. This epigenetic modification stimulated stranded RNA of ∼22 nucleotides in length. It is complementary the recruitment of MLL1, a trithorax group (TrxG) protein, to the site of the 3′ untranslated region of the target gene mRNA and increased H3K4me3 that augmented transcriptional activity and binds through sequence-specific base pairing. The latest from the Ink4a/Arf locus. During β-cell regeneration, increased research shows that microRNA is closely related to pancreatic Bmi-1 binding coincided with decreased H3K4me3, resulting development, insulin secretion and insulin resistance, and may

Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 August 2020 | Volume 8 | Article 748 Xu et al. The Epigenetic Regulation in Pancreatic β Cell

play an essential role in the pathogenesis of diabetes (Table 4) TABLE 4 | miRNAs in the differentiation and function of pancreatic β cells. (Gilbert and Liu, 2012). miRNAs Targets Functions miR-375 is the first miRNA found to be involved in glucose metabolism and is highly expressed in pancreatic islets. Poy miR-7 GATA6, PDX6 Regulate development and function of et al. found that inhibiting endogenous miR-375 not only endocrine pancreas (Joglekar et al., 2011; reduces insulin secretion but also affects the proliferation of Latreille et al., 2014) pancreatic β cells (Joglekar et al., 2007). In mice lacking miR- Pfn2, Wipf2 Regulate insulin secretion (Latreille et al., 375 (mi-375 knockout), the number of pancreatic islet alpha 2014) cells increased, and the number of pancreatic β cell clusters Mknk2, Mknk1, Regulate the proliferation and differentiation eIF4E, S6k1 of islet cells (Wang et al., 2013) decreased, leading to increased blood glucose levels (Xu et al., 2009). PDX1 is one of the confirmed target genes of miR-375, miR-375 Mtpn, p38 Promote the proliferation, differentiation and MAPK, PI3K transdifferentiation of β cells (Ozcan, 2014) and overexpression of miR-375 can downregulate PDX1 in the CADM1, CAV1 Regulate pancreatic development PI3-K pathway to reduce the expression of insulin genes and (Kloosterman et al., 2007) inhibit the proliferation of pancreatic β cells (Garikipati et al., PDK1 Regulate the biosynthesis of insulin (Tang 2017). Mir-375 also plays a regulatory role in the differentiation et al., 2007; El Ouaamari et al., 2008) of islet cells. By analyzing the dynamic profile of the miRNA miR-9 Onecut-2, SIRT1 Inhibit insulin secretion (Plaisance et al., expression during the differentiation of iPSCs from hESCs, it 2006) was found that the expression of miR-375 increased at the early miR-96 NOC2 Increase granuphilin/Slp4 and decrease stage of iPSC differentiation and then decreased. In addition, Noc2 miR-375 has a negative regulatory effect on the specific target miR-15b, Ngn3 Regulate pancreatic development (Joglekar miR-16, et al., 2007) genes HNFfl and Sox9 in the development of islet embryos to miR-195, promote the differentiation and maturation of pancreatic β cells miR-106b (Poy et al., 2004). miR-21 PTEN, PI3K, Regulate pancreatic development (Ozcan, Krek et al. found that miR-124a is highly expressed in MIN6 AKT 2014) and has a synergistic effect with miR-375, which has been shown miR-34a VAMP2, SNARE Decrease insulin production (Roggli et al., to jointly inhibit the myotrophin expression (Krek et al., 2005). 2010) FoxA2 is a well-defined miR-124a target gene that has important miR-124a, PDX-1, Kir6.2, Promote the differentiation of β cells regulatory effects on pancreatic β cell differentiation and insulin let-7b SUR1, TGF-β / (Ozcan, 2014) TGF-β-R secretion. Baroukh et al. found that miR-124a binds the FoxA2 pathway ′ mRNA 3 UTR region, which can directly regulate the protein miR-106b, Ngn3 Promote the formation of endocrine expression level of FoxA2 in pancreatic β cells (Baroukh et al., miR-15a, precursor cells (Joglekar et al., 2007) 2007). As the expression level of the FoxA2 gene decreases, miR-15b, the expression of downstream target genes increases, including miR-16, PDX1, Kir6.2 and sulfonylurea receptor 1 (SUR1). miR-195 NeuroD1 miR-7 is the most abundant miRNA expressed in the islets miR-19b, Promotes the differentiation of pancreatic miR-17- precursor cells into endocrine precursor of humans and rats (Correa-Medina et al., 2009). It is highly 92/217 cells (Taylor et al., 2013) expressed in the pancreatic islets of humans at 14–18 weeks miR-18a, Ptf1a Promotes the differentiation of pancreatic of gestational age, a time at which the secretion of endocrine miR-145, precursor cells into endocrine precursor hormones by islets increases exponentially. Similar to miR-375, miR-495 cells (Krapp et al., 1998) miR-7 is upregulated during the development and differentiation miR-29 AMPK Inhibit pancreatic cell proliferation, promote of human islets (Correa-Medina et al., 2009). Nieto et al. apoptosis and autophagy (Pullen et al., 2011) established a mouse model in which miR-7 was knocked out miR-145, Oct4, Sox2, Klf4 Promote the differentiation of stem cells at embryonic E10.5. The synthesis of mouse insulin in the miR-21 into human cells (Xu et al., 2009) embryo was reduced and the number of pancreatic β cells was miR-290-295, Sox2 Promote the differentiation of stem cells reduced (Nieto et al., 2012). MiR-7 is an important part of miR-302 into human cells (Marson et al., 2008) rapamycin complex 1 (mTORC1) and its down-stream effector miR-182 Rasa1, Grb2 Maintain the stability of β cells S6K1 signaling, and the inhibition of mir-7a can activate mTOR (Melkman-Zehavi et al., 2011) signal to promote the proliferation of primary pancreatic β cells miR-23b Hes1 Promote the differentiation of pancreatic in adult mice (Wang et al., 2013). endocrine cells (Ozcan, 2014) miR-19 is an important member of the miR-17-92 gene cluster, miR-15a UCP2 Regulate the biosynthesis of insulin (Sun et al., 2011) which is highly enriched in pancreatic precursor cells, while its Ngn3 Regulate endocrine pancreas development expression in pancreatic endocrine precursor cells shows a sharp and regeneration (Joglekar et al., 2007) downward trend in differentiated cells. This suggests that miR- miR-30 Vimentin, Snail1, Regulate the proliferation and development 19b is involved in the differentiation of pancreatic precursor Rfx6 of β cells (Liao et al., 2013) cells into endocrine precursor cells. Further research shows that NeuroD is the target gene of miR-19b and that miR-19b can (Continued)

Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 August 2020 | Volume 8 | Article 748 Xu et al. The Epigenetic Regulation in Pancreatic β Cell

TABLE 4 | Continued TABLE 5 | lncRNAs and circRNAs in the differentiation and function of pancreatic β cells. miRNAs Targets Functions LncRNAs or Location Targets Functions Map4k4 Regulate the biosynthesis of insulin (Zhao circRNAs et al., 2012) NeuroD Regulate insulin secretion (Kim et al., 2013) KCNQ1 11p15.5 CDKN1C Regulate the proliferation and development of β cells (Avrahami miR-33a ABCA1 Regulate insulin secretion (Roggli et al., et al., 2014) 2010) H19 11p15.5 IGF2 Regulate insulin secretion (Hark et al., miR-130b SOX9, PDX6 Maintain the number of pancreatic 2000; Schoenherr et al., 2003) progenitor cells (Rosero et al., 2010) MEG3 14q32 DLKl, RTL1 Regulate the biosynthesis of insulin miR-184 Argonaute2 Regulate compensatory amplification of β (Kameswaran et al., 2014) cells (Tattikota et al., 2014) PLUT 13q12.2 PDX1 Regulate the development of β cells miR-200 c-Maf, Fog2, Regulate pancreatic differentiation (Liao and insulin secretion (Yin et al., 2015) Zeb, Sox17 et al., 2013) TUG1 22q12.2 / Regulate the biosynthesis and miR-218, HNF6, Onecut2 Regulate pancreatic development (Simion secretion of insulin (Yin et al., 2015) miR-495 et al., 2010) H1-LNC25/ 20q12 Glis Regulate the development of β cells miR-342 Foxa2, MafB, Regulate the proliferation and development LINC01370 (Ravassard et al., 2011) GATA4 of β cells (Kloosterman et al., 2007) CDR1as Xq27.1 miR-7 Promote development and function of miR-382 ISL1 Regulate the differentiation and function of endocrine pancreas; endocrine cells (Rosero et al., 2010) promote insulin secretion (Hansen Pfn2, profilin 2; Wipf2, WAS/WASL interacting protein family member 2; eIF4E, eukaryotic et al., 2013) translation initiation factor 4E; S6k1, Ribosomal protein S6 kinase 1; Mapkap1, MAPK circHIPK3 11p13 miR-338-3p, Promote the proliferation and associated protein 1; Mknk1, MAP kinase interacting serine/threonine kinase 1; Mknk2, miR-124-3p development of β cells; MAP kinase; interacting serine/threonine kinase 2; SIRT1, silent information silent promote insulin secretion (Stoll et al., informationregulation 2 homolog-1; Onecut-2: one cut homeobox 2; UCP2, uncoupling 2018) protein 2; Ngn3, neurogenin 3; Ptf1a, pancreas associated transcription factor 1a; NeuroD1, neurogenic differentiation 1; SOX6, SRY-boxtranscription factor 6; SOX17, SRY- box transcription factor 17; MCT1, monocarboxylate transporter 1; Mcl-1, myeloid cell leukemia-1; Snail1, snail family transcriptional repressor 1; Map4k4, mitogen-activated protein kinase kinase kinase kinase 4; Abca1, ATP binding cassette subfamily A member functions, or can be used as a structural component to form 1; Zeb, zinc finger E-box binding homeobox; HNF6, hepatocyte nuclear factors 6; MafB, v-maf musculoaponeurotic fibrosarcomaoncogene homolog B; CADM1, cell adhesion nucleic acid-protein complexes and bind to the promoter molecule 1; PDK1, pyruvate dehydrogenase kinase 1; ISL1, islet1. to regulate target gene transcription. Some lncRNAs serve as molecular sponges to bind miRNAs, reducing miRNA bioavailability and regulate the expression of target genes (Table 5)(Guay et al., 2012; Xin et al., 2017). bind to the 3′-UTR sequence of NeuroD, which inhibits its expression and then downregulates the expression of the insulin KCNQ1 Gene gene (Yanaihara et al., 2006). miR-9 can regulate Sirtl gene expression to regulate insulin The KCNQ1 gene is one of the members of the voltage- secretion in pancreatic β cells (Ramachandran et al., 2011; Xu dependent potassium channel family (Splawski et al., 1998). The et al., 2013). In addition, miRNAs induced by inflammatory gene is located at 11p15.5, nearing the imprinting site H19- factors can mediate the death of pancreatic β cells, leading to IGF2 (Hussain et al., 2005). This imprinting region includes insulin secretion defects. For example, the inflammatory factors some conserved maternally expressed protein-coding genes, such TNFα and IL-1 in chronic inflammation can upregulate the as Cyclin-dependent kinase inhibitor 1C (CDKN1C) and the expression of miR-21, miR-34a and miR-146, and these miRNAs paternal lncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) in turn regulate the differentiation of pancreatic β cells and (Monk et al., 2006). Loss of this imprinting site can lead to the insulin secretion. In addition, miR-30d, miR-15a, miR-24, inhibition of CDKN1C, causing β cells to re-enter the cell cycle miR-148a, and miR-182 also participate in the regulation of the and resulting in increased insulin levels and decreased blood expression of the insulin genes in pancreatic β cells (Melkman- glucose levels (Avrahami et al., 2014). Zehavi et al., 2011). Unoki et al. applied the genome-wide associate study (GWAS) method to conduct a comprehensive screening of 207,097 single nucleotide polymorphisms (SNPs) and analyzed the genetic The Role of lncRNAs in β Cell polymorphisms of the T2DM group and the healthy control Differentiation and Function group (Unoki et al., 2008). The risk of T2DM is significantly LncRNA is a type of noncoding RNA with a transcription related to 6 SNPs (rs2237897, rs2237896, rs2299620, rs2237895, length greater than 200 nucleotides. Studies have found that rs227892, and rs163171), which can triple the risk of T2DM lncRNAs are involved in the pathophysiological processes of (Travers et al., 2013). The risk allele of homozygous rs2237895 diabetes, tumors, nervous system diseases and other diseases in fetal samples increased slightly in the KvDMR region but (Liu et al., 2016; Meng et al., 2017; Qi et al., 2017) LncRNA was not observed in adults because KCNQ1 and KCNQ1OT1 can combine with proteins to regulate downstream protein are expressed in only the fetus (Travers et al., 2013). These

Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 August 2020 | Volume 8 | Article 748 Xu et al. The Epigenetic Regulation in Pancreatic β Cell results indicate that the risk allele may be in the islets. In metabolism disease characterized by abnormal insulin secretion the early stages of development, this study proposed a model by pancreatic β cells and hypoglycaemia (Ohlsson et al., 1993). in which the risk allele of the rs2237895 SNP led to an ICR hypermethylation can lead to a decrease in H19 expression increase in KvDMR methylation, which resulted in a decrease in and an increase in IGF2 expression. Devedjian and Fournet et al. KCNQ1OT1 expression. However, KCNQ1OT1 expression did found that although IGF2 was overexpressed in mouse FoCHI not differ significantly between the T2DM and control groups. In model, expression in the human FoCHI model was different; contrast, the transcription level of KCNQ1OT1 was significantly however, H19 transcription was consistently downregulated, increased in the islet of the pancreas in T2DM, which was indicating that H19 has a very important regulatory role in associated with an overall decrease in CpG methylation of the inhibiting the proliferation of pancreatic β cells (Devedjian et al., KCNQ1 gene (Moran et al., 2012). Therefore, the pathological 2000; Fournet et al., 2001). This type of ICR hypermethylation interpretation of the variants in this region has always been with decreased expression of H19 has also been reported in a contradiction and challenge. However, the regulation of this nephroblastoma. As mentioned previously, the lncRNA H19 site and lncRNA KCNQ1OT1 is still closely related to the regulates β cell function and cell proliferation, and its level can pathogenesis of β cell biology and T2DM, and the mechanism be adjusted independently or indirectly by adjusting the level needs further study. of IGF2.

H19/IGF2 Gene Locus DLK1/MEG3 Gene Cluster The H19/IGF2 gene locus is located on chromosome 11p15.5 DLK1-MEG3 gene cluster is located on chromosome 14q32 adjacent to KCNQ1, and includes the IGF2 gene expressed in humans (chromosome 12 in mice) and includes paternally by the male parent and the H19 lncRNA expressed by the expressed protein-coding genes, such as DLK1, RTL1, and DIO3, female parent (Brannan et al., 1990). The former is essential and maternally expressed non-coding RNAs, such as MEG3, for early embryonic development, while the latter can inhibit miRNAs, and snoRNAs. DLK1 is expressed in many embryonic tumors (Hao et al., 1993). The imprinting control region (ICR) is tissues and is a negative regulator of adipocyte differentiation. located between these two genes. On the maternal chromosome, It is highly expressed in human and mouse β cells (Appelbe the ICR is unmethylated and binds to the transcription factor et al., 2013). This cluster is strictly regulated by the ICR, which CCCTC (CTCF). This interaction has an insulator function consists of two paternal differentially methylated regions (DMR). to block the downstream enhancer from binding to the IGF2 One region called IG-DMR, which is the primary ICR, is located gene promoter so that the downstream enhancer instead acts at 13kb upstream of the MEG3 transcription start site. The on the H19 promoter and promotes H19 expression. Because other region overlaps with the MEG3 multi-cis-trans acting the ICR is methylated on the chromosome, it cannot bind to transcription site promoter, called the MEG3-DMR. Deletion of the CTCF transcription factor and cannot act as an insulator. this imprinted area can lead to maternal or paternal diploidy Therefore, the downstream enhancer cannot bind to the H19 of chromosome 14 (uniparental disomy, UPD), thereby causing gene promoter, but instead binds only to the IGF2 gene promoter. various severe developmental disorders. Thus, the downstream enhancer inhibits the expression of the A recent study revealed that the non-coding RNA of the H19 gene and promotes the expression of IGF2 (Hark et al., 2000; DLK1/MEG3 imprinting site is downregulated in the islets Schoenherr et al., 2003). of T2DM patients, but the specific mechanism is unclear Loss of ICR methylation at the H19/IGF2 locus inhibits IGF2 (Kameswaran et al., 2014). Increased methylation of the expression, which can cause syndromes of short stature and MEG3/DMR and decreased expression of MEG3 can also be low birth weight in rodent models and humans; one example is observed in some cancers, such as pituitary cancer, renal Silver-Russell syndrome, which is a characteristic developmental cell carcinoma, and multiple myeloma. In vitro experiments disorder characterized by retardation of intrauterine and have further revealed that MEG3 can exert tumor suppressive postpartum growth (DeChiara et al., 1990). To ensure the effects by activating the p53 gene; these functions are partially development of important organs, metabolic processes in fat, dependent on the secondary structure of the MEG3 RNA. In skeletal muscle and other peripheral tissues can undergo addition, the decrease in MEG3 expression in T2DM may be permanent changes and lead to insulin resistance. This closely related to increased methylation of the MEG3/DMR. observation is called the “thrifty phenotype hypothesis.” However, Ding et al. found that impaired glucose tolerance in LncRNA PLUT the progeny of mice with GDM is accompanied by H19 ICR The lncRNA Pdx1-associated lncRNA upregulator of hypermethylation in islets (Ding et al., 2012). This contradictory transcription (PLUT) is abundantly expressed in the nucleus of finding may be caused by exposure of the developing fetus to islet β cells, and its enhancer region can bind to the promoter different nutritional states or by different exposure times. The of the key pancreatic β cell transcription factor PDX1 to above studies show that the H19/IGF2 gene locus can respond to participate in regulating pancreatic islet β cell transcription and the intrauterine environment and can be used to predict future differentiation processes (Yin et al., 2015). PDX1 can induce metabolic complications. endoderm cells to develop and mature into pancreatic cells and Hypermethylation of the H19/IGF2 ICR can be found in some can further induce their differentiation into insulin-secreting patients with Beckwith-Wiedemann syndrome (BWS) and focal cells. On the other hand, PDX1 is also involved in mediating congenital hyperinsulinaemia (FoCHI); the latter is a glucose insulin transcription factors and glucose transport, which

Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 August 2020 | Volume 8 | Article 748 Xu et al. The Epigenetic Regulation in Pancreatic β Cell is important in the development of the pancreas and in the (ciRNAs), and exonic/intronic circRNAs (EIciRNAs) (Stoll et al., regulation of insulin secretion. Therefore, loss of the lncRNA 2018; Li R. et al., 2019). With the rapid development of high- PLUT, leads to downregulation of PDX1 expression, will lead to throughput sequencing and other biotechnologies, numerous abnormal development of the pancreas. circRNAs have been found in various organisms, such as nematodes, fruit flies, mice, macaques, and humans. Further LncRNA TUG1 studies have revealed that circRNAs play important roles in the Pancreatic β cells are the main insulin-producing cells, but they occurrence and development of cancer, nervous system diseases, are also regulated by insulin and dysfunction of these cells is a cardiovascular diseases, and metabolic diseases (Wang et al., key process in the development of T2DM. Studies have shown 2019; Xiao, 2020). that lncRNAs are involved in regulating the secretory function Hansen et al. believed that CDR1as could negatively regulate of pancreatic β cells. The lncRNA TUG1 is expressed in β cells. miR-7 through sponging miR-7, suppress the inhibitory effect of Yin et al. silenced the TUG1 gene in adult non-obese diabetic miR-7 on the mTOR signaling pathway, and stimulate pancreatic (NOD) mice via siRNA transfection and found that islet β β cell proliferation and insulin secretion (Hansen et al., 2013). cell synthesis and insulin secretion were significantly reduced, The study showed that circHIPK3 was downregulated in the islets while islet β cell apoptosis was increased (Yin et al., 2015). This of diabetic model mice which inhibited β cell proliferation and pattern suggests that TUG1 may be involved in maintaining insulin secretion. The mechanism may involve the competitive islet β cell function and may play an important role in glucose interaction of circHIPK3 with miR-338-3p and miR-124-3p to metabolism homeostasis. High-glucose conditions further inhibit regulate the key genes in the development, proliferation and the expression of the TUG1 gene in pancreatic β cells, causing a function of pancreatic β cells, such as glucose transporter 2 vicious cycle and triggering diabetes. (Glut2) and protein kinase B1 (PKB1) (Stoll et al., 2018). Zhao et al. found 489 circRNAs differently expressed in the peripheral lncRNA H1/LNC25 blood of T2DM patients. They further verified that circRNA- LncRNA H1/LNC25 is a multifunctional exon transcript cluster 0054633 (hsa_circ_0054633) could be used as a diagnostic marker that is highly expressed in pancreatic β cells and participates in for T2DM (Zhao et al., 2017). Hsa_circ_0054633 is not only the regulation of insulin secretion. Studies have shown that the involved in biological processes such as cell cycle and mitotic expression of lncRNA H1/LNC25 in pancreatic β cells of diabetic arrest but is also closely related to the pathogenesis of diabetes patients is significantly reduced (Ravassard et al., 2011). Moran mellitus (Werfel et al., 2016; Zhao et al., 2017). By collecting et al. found that the expression of Glis family zinc finger 3 (Glis3) peripheral blood of patients with T2DM, coronary heart disease, mRNA was significantly reduced upon the inhibition of lncRNA T2DM complicated with coronary heart disease and health H1/LNC25 expression in human pancreatic β cells (Moran et al., group, the results of circRNA microarray showed that HAS-cirC- 2012). Glis3 is a member of the Glis-like zinc finger family and 11783-2 was closely related to T2DM and coronary artery disease encodes an insulin transcription factor. Loss of Glis3 expression (Salzman et al., 2013). causes the insulin secretion of pancreatic β cells to decrease Although accumulating studies have indicated that circRNAs (Nogueira et al., 2013). Therefore, it can be speculated that play key regulatory roles in the occurrence and development of the lncRNA H1/LNC25 affects glucose metabolism by affecting diabetes, the more specific biological functions and molecular the expression of Glis3, which in turn leads to the occurrence mechanisms need to be further revealed. of diabetes. In summary, lncRNAs, which are key factors regulating the CONCLUSION homeostasis of glucose metabolism homeostasis, play important roles in the developmental regulation and function of islet β Epigenetic regulation of pancreatic islets is of major interest cells development and biological function. A single lncRNA can to understand how endocrine cells differentiate, proliferate, act through multiple pathways, and multiple lncRNAs can also establish, and maintain their mature identity and function. The act together within the same pathway. Although the current development process of β cells from multipotent progenitors understanding of lncRNAs is insufficient, detection detecting the can be successfully recapitulated only partially in vitro to expression of specific lncRNAs expression in tissues or body generate pancreatic progenitors and functional β cells in cell fluids may be a new means of approach to predicting the risk replacement therapy for treating diabetes. Thus, the epigenomic of diabetes. The discovery of additional lncRNAs will provide us study of pancreatic islets cells has become a necessity to with more comprehensive ways to improve models for research overcome the barriers that restrict lineage plasticity and is an on glucose metabolism homeostasis research models. exciting prospect for the future development of successful β-cell differentiation strategies (Balaji et al., 2018; Lv and Huang, Roles of Circular RNAs (circRNAs) in β Cell 2019; Scarpa, 2019; Gong and Jiang, 2020). Dissecting the Differentiation and Function epigenetic components of islet cell-specific transcriptional CircRNAs are closed, circular, non-coding RNAs that do not have networks would contribute to induce and maintain appropriate 5’ caps or 3′ poly(A) tails, are not easily degraded by exonucleases, endocrine lineage commitment from embryonic stem cells and have the characteristics of structural stability, tissue or induced pluripotent cells. Knowledge and manipulation of specificity, and evolutionary conservation (Stoll et al., 2018). the PcG-repression and derepression programs of pancreatic CircRNAs can be divided into three categories according to progenitors and β cells would provide benchmarks to direct their sources: exonic circRNAs (e-circRNAs), intronic circRNAs β cell differentiation protocols. Epigenetic manipulations

Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 August 2020 | Volume 8 | Article 748 Xu et al. The Epigenetic Regulation in Pancreatic β Cell demonstrated the importance of DNA methylation and histone interpreted the data and wrote the manuscript. All authors read modifications in maintaining β-cell identity and driving islet and approved the final manuscript. cell differentiation. They have underscored the potential of using reprogramming methods to change identity or regain FUNDING plasticity of adult endocrine cells and generate novel sources of islet cells. It is also expected that the better understanding This project was supported by grants from the National of the epigenetic mechanisms leading to enhanced β-cell Science Foundation of China (number 8187120707), the mass, or to β-cell proliferation and function defects during Produce-Learn-Research Projects of Shanghai University of diabetes, allows identification of novel therapeutic targets. A Medicine & Health Sciences (number B1-0200-19-311144), detailed understanding of the specialized functions of epigenetic and Nutrition and food safety micro-specialty construction regulators in healthy and diabetic islets, together with the project of Shanghai University of Medicine & Health Sciences generation of specific compounds targeting these components, (JG 2005-g1-01). may accelerate development of strategies for islet replacement or regeneration therapies for prevention and treatment of diabetes. ACKNOWLEDGMENTS

This manuscript was edited for proper English language, AUTHOR CONTRIBUTIONS grammar, punctuation, spelling, and overall style by one or more of the highly qualified native English-speaking editors at FX and JL conceived and designed the study. FX and LN American Journal Experts (Certificate Verification Key: AF0F- performed the data collection and analysis. FX and LC 828C-348D-0712-7E8P).

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