The hallmark of diabetes mellitus is glucose imbalance caused by the loss and/or dysfunction of the pancreatic ?-cell. Current research is focused on retaining/replacing the function of the pancreatic ?-cell mass. The molecular mechanisms that govern islet ?-cell development, maintenance, and function often involve the actions of transcription factors. The Stein lab and others have identified the large Maf proteins, MafA and MafB, as crucial regulators of ?-cell development and function. These transcription factors bind to cis-acting enhancer sequences as either homodimers (MafA2, MafB2) or heterodimers (MafA/B) and subsequently recruit coregulatory trans-acting factors. These coregulators modulate transcription through reorganization of nucleosomes and modification of histones and the basal transcription machinery. Rodents exclusively express MafA in mature ?-cells, while humans express both MafA and MafB. Unfortunately, little information exists concerning the function of MafA/B compared to MafA2 and MafB2 in adult human ?-cells. I have generated a mouse model (MafBtg) that misexpresses MafB in the mature ?-cell, mimicking the human adult ?-cell. I have validated the sequence integrity, molecular weight, and distribution pattern of the MafB transgene, as well as established the existence of the MafA/B heterodimer in MafBtg islets.
In Aim 1, I will examine the molecular consequences of MafA/B on islet ?-cell activity. Preliminary data suggests that the roles of MafA2 and MafA/B overlap, given that MafBtg animals exhibit normal glucose clearance when challenged with glucose. Gene expression analysis (qPCR), MafA/B promoter occupancy (re-ChIP), and perifusion analysis (GSIS) will be performed in MafBtg islets to further examine the role of MafA/MafB. Although MafA and MafB share similar structures, they exhibit distinct functional characteristics.
In Aim 2, I will investigate similarities and differences between MafA2 and MafB2 in adult ?-cells by crossing MafBtg animals with animals devoid of MafA expression in ?-cells (MafA??), a model that mimics the juvenile human ?-cell. Preliminary results have revealed that MafB does not rescue glucose intolerance in the MafA?? background. Techniques used in Aim 1 will be utilized to delineate the role of MafB2 in comparison to MafA2 and MafA/B. MafA and MafB transcriptional activities may not be equivalent due to a divergent recruitment of distinct coregulators. Coregulators of MafA/B and MafB2 will be identified in Aim 3 by using a reversible "in-cell" cross-link immunoprecipitation (Re-CLIP), mass spectrometry (MS) strategy in EndoC-?H1 cells, a newly characterized human ?-cell line that express both MafA and MafB. Candidate coregulators will be tested in co-IP, re-ChIP and proximity ligation assays (PLAs) for validation. Collectively, this proposal aims to identify the significance of MafB2 and MafA/B in islet functions and coregulators essential for their transcriptional regulation. Knowledge gained from this proposal will significantly enhance our understanding of the molecular details underlying the human ?-cell and will be useful toward the efforts of generating/retaining ?-cells for the treatment of diabetes.
In this proposal, the function of the MafA/B heterodimer that exists in human adult -cells will be tested in a mouse model that mimics human -cells (MafBtg) and the human -cell line (EndoC-H1). Pancreatic islet - cells and EndoC-H1 cells will be subjected to molecular- and physiological-based assays to delineate distinctions in gene targets and coregulators between MafA2 (found in rodent adult -cells), MafB2 (found in human juvenile -cells) and MafA/B (found in human adult -cells). Together, these data will demonstrate the importance MafA/B and MafB2 and the mechanism of action by which these factors regulate and maintain human adult -cell function.
|Banerjee, Ronadip R; Cyphert, Holly A; Walker, Emily M et al. (2016) Gestational Diabetes Mellitus From Inactivation of Prolactin Receptor and MafB in Islet Î²-Cells. Diabetes 65:2331-41|
|Scoville, David W; Cyphert, Holly A; Liao, Lan et al. (2015) MLL3 and MLL4 Methyltransferases Bind to the MAFA and MAFB Transcription Factors to Regulate Islet Î²-Cell Function. Diabetes 64:3772-83|