Accepted Manuscript PDX1 and ISL1 differentially coordinate with epigenetic modifications to regulate insulin gene expression in varied glucose concentrations Weiping Wang, Qiong Shi, Ting Guo, Zhe Yang, Zhuqing Jia, Ping Chen, Chunyan Zhou PII: S0303-7207(16)30067-3 DOI: 10.1016/j.mce.2016.03.019 Reference: MCE 9454 To appear in: Molecular and Cellular Endocrinology Received Date: 1 November 2015 Revised Date: 26 February 2016 Accepted Date: 15 March 2016 Please cite this article as: Wang, W., Shi, Q., Guo, T., Yang, Z., Jia, Z., Chen, P., Zhou, C., PDX1 and ISL1 differentially coordinate with epigenetic modifications to regulate insulin gene expression in varied glucose concentrations, Molecular and Cellular Endocrinology (2016), doi: 10.1016/j.mce.2016.03.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Regular paper PDX1 and ISL1 differentially coordinate with epigenetic modifications to regulate insulin gene expression in varied glucose concentrations Weiping Wang, Qiong Shi, Ting Guo, Zhe Yang, Zhuqing Jia, Ping Chen, Chunyan Zhou* Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences; Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function; Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China; Peking University, 38 Xue Yuan Road, Beijing 100191, China *Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, MANUSCRIPTPeking University, 38 Xue Yuan Road, Beijing 100191, China, Phone: 86-10-82802417. Fax: 86-10-62015582. E-mail: [email protected] ACCEPTED 1 ACCEPTED MANUSCRIPT ABSTRACT The mechanism of insulin gene transcription control in response to glucose concentration is poorly defined. The islet-restricted transcription factors PDX1 and ISL1 interact with BETA2, activating insulin gene expression. However, their contribution and hierarchical organization in insulin expression control based on glucose concentration remain unknown. We investigated PDX1 and ISL1 regulation of insulin gene expression in pancreatic β cells cultured in normal (5 mM/L) and high (25 mM/L) glucose conditions. ISL1 interacted with BETA2 to maintain basic insulin gene transcriptional activity under normal glucose. The ISL1-recruited cofactors SET9 and JMJD3 facilitated insulin gene histone modifications under normal glucose. In high-glucose concentrations, PDX1 formed a complex with BETA2 to enhance insulin gene expression. PDX1 also recruited SET9 and JMJD3 to promote the activation of histone modulation on the insulin promoter. This is the first evidence transcription factors orchestrate epigenetic modifications to control insulin gene expression based on glucose concentration. MANUSCRIPT Keywords: Insulin, PDX1, ISL1, Glucose, SET9, JMJD3 ACCEPTED 2 ACCEPTED MANUSCRIPT 1. Introduction Insulin is one of the critical hormones for regulating blood glucose levels and is produced exclusively by pancreatic β cells. The insulin ( INS ) gene was first cloned and sequenced in 1980, opening up a new field of research on the mechanisms controlling its expression (Bell, Pictet, Rutter et al., 1980). To date, more than 40 regulatory transcription factors of insulin gene expression have been identified (Melloul, Marshak and Cerasi, 2002,Hay and Docherty, 2006). However, the circumstances under which these factors play their roles in regulating insulin gene transcription remain unclear. The islet-restricted transcription factors PDX1 (pancreatic and duodenal homeobox 1), ISL1 (insulin gene enhancer binding protein, islet factor 1), and BETA2 (beta cell E-box transcription factor 2) bind to the A3/A4 and E2 boxes located between 0 and -410 bp upstream of the transcription start site in the rat insulin I promoter (RIP1) (Le Lay and Stein, 2006,Karlsson, Thor, Norberg et al., 1990,Ohlsson, Karlsson and Edlund, 1993). MANUSCRIPTThis cis -acting regulatory sequence is essential for glucose regulation of insulin gene expression (Melloul, Ben-Neriah and Cerasi, 1993); glucose is the central regulator of β cell function and insulin gene expression (Nielsen, Welsh, Casadaban et al., 1985,Evans-Molina, Garmey, Ketchum et al., 2007). PDX1, a master regulator of insulin gene transcription, has been implicated in glucose regulation of insulin gene transcription. PDX1 interacts with histone acetyltransferase p300 to hyperacetylate histone H4 on the insulin gene promoter in response to high glucose concentrations (Mosley, Corbett and Ozcan, 2004). In addition to PDX1, ISL1, a LIM homeodomain protein, also binds to the A3/4 box ofACCEPTED the insulin promoter. As a key transcription factor, ISL1 is not only involved in insulin gene regulation (Peng, Wang, Meng et al., 2005,Zhang, Wang, Guo et al., 2009), but also in cell fate specification and embryonic development (Thor, Ericson, Brannstrom et al., 1991,Wilfinger, Arkhipova and Meyer, 2013). However, whether ISL1 is involved in glucose regulation of insulin gene expression remains unclear. 3 ACCEPTED MANUSCRIPT Intriguingly, previous research has revealed that both PDX1 and ISL1 interact with BETA2, a bHLH transcription factor that binds to the E2 box of the insulin promoter, to activate insulin gene expression (Zhang et al., 2009,Ohneda, Mirmira, Wang et al., 2000). However, no light has been shed on the detailed mechanism underlying how PDX1 and ISL1 influence insulin gene expression under different physical conditions, such as blood glucose levels. Whether the activation of insulin gene expression involves the occurrence of a similar or differing activation effect, or even a competitive effect, remains to be elucidated. In the present study, we explored the molecular mechanism underlying the function of PDX1 and ISL1 in regulating insulin gene expression in response to environmental glucose stimuli. Recent studies have reported increasingly complicated modes of insulin gene regulation in pancreatic islet β cells, which not only involve traditional transcription regulation such as protein–protein and protein–DNA interactions, but also epigenetic modification (Moon, Han, Kim et al., 2014,Qiu, Guo, Huang et al., 2002,Ramalingam, Lu, Hudmon et al., 2014). Gain of histone H3 trimethylated at lysine 4 (H3K4me3) and loss of H3K27me3 on the gene promotersMANUSCRIPT are essential for inducing heterochromatin to euchromatin formation and gene transcription activity (Del Rizzo and Trievel, 2014,Estaras, Fueyo, Akizu et al., 2013). SET9 is an islet-enriched histone H3 K4–specific methyltransferase that enhances PDX1 transcription activity on insulin gene regulation (Francis, Chakrabarti, Garmey et al., 2005). Our recent research has also shown that SET9 functions not only as a histone methyltransferase that increases histone H3 K4 trimethylation on the cyclin D1 promoter region, but also as an adaptor to bridge ISL1 and PDX1 in pancreatic islet β cells (Yang, Zhang, Lu et al., 2015). As a key H3K27me3 demethylase, JMJD3 plays an important role in regulating geneACCEPTED expression in response to environmental signaling stimuli (He, Kim, Kim et al., 2015). We have identified a number of ISL1- or PDX1-interacting proteins by mass spectrometry in pancreatic islet β cells (Yang et al., 2015). Among these factors, we found that SET9 and JMJD3 could be potential candidate epigenetic modifiers. Hence, we investigated whether ISL1 or PDX1 could affect insulin gene transcription in different glucose concentrations not only through traditional 4 ACCEPTED MANUSCRIPT transcription regulation, but also in association with epigenetic modification. In the present study, we demonstrate that ISL1 interacts with BETA2 to maintain basic insulin gene transcriptional activity in normal glucose conditions. In high-glucose conditions, PDX1 forms a complex with BETA2 to enhance insulin gene expression. As cofactors of ISL1 or PDX1, SET9 and JMJD3 facilitate insulin gene histone modification under different glucose conditions. These observations delineate a novel and precise insulin gene regulation pattern in response to glucose signals. Our study provides the first evidence that transcription factors orchestrate epigenetic modifications for proper control of insulin gene expression in response to environmental signaling stimuli. 2. Materials and Methods 2.1 Diabetes animal model and rat pancreatic islet isolation The animal experiments were performed in accordance with the ethical principles and guidelines for scientific experiments on animals of the Swiss Academy of Medical Sciences (1995). The Animal Care andMANUSCRIPT Use Committee of Peking University approved all protocols (LA 2010-066). Db/db mice (14–16 weeks old), which are homozygous for the db gene and exhibit an obese and diabetic phenotype, were used for the type 2 diabetes animal model. Db/m mice, which are heterozygous for the db gene and exhibit a non-diabetic, normal phenotype, were used as controls to the db/db mice. The mice were anesthetized with 5 mg/100 g body weight sodium pentobarbital, and the pancreatic tissues were removed. RNA was prepared from these tissues and used for real-time