(30 lines) Diabetes mellitus is characterized by glucose imbalance due to a loss and/or dysfunction of pancreatic islet cells. With the goal of retaining or restoring islet cell function, many groups have determined the roles of transcription factors in ? and ? cell development and activity. The Stein lab and others identified and characterized the transcription factors MafA and MafB, which recognize and bind to DNA as homo- or heterodimers. In rodents, MafB is expressed in immature ? and ? cells but only in mature ? cells, and functions in islets primarily during development, while MafA expression is exclusive to insulin+ cells and is required for adult ? cell activity. In humans, MAFB is also expressed prior to MAFA; however, its expression is maintained in ? and ? cells, an important distinction from mouse. Furthermore, both MAFA and MAFB expression are lost in diabetic islets; however, little is known about the regulation of ? and ? cell gene expression by MAFB2 and MAFA/B in humans. In addition, few trans-acting coregulators, which positively or negatively impact the transcriptional state of DNA, have been identified for islet cell transcription factors. The human ? cell line, EndoC?H2, emulates human ? cell development by expressing MAFB prior to Cre recombinase mediated excision of the immortalization factors hTERT and SV40 Tag. Following Cre mediated excision, MAFA levels are elevated parallel to the induction of mature ? cell-like properties such as increased Insulin expression and secretion. Of note, the significant increase in transcription factor expression is specific to MAFA indicating that MAFA may drive ? cell maturation.
In Aim 1 I will use siRNA knockdown of MAFA or MAFB and RNA-Seq to define the regulatory roles of MAFB2 in immature ? cells and the heterodimeric MAFA/MAFB activator in adult. Lentivirus will also be used to deliver siRNA against MAFB or MAFA in human islets followed by qRT-PCR to confirm gene expression changes determined in EndoC?H2 or EndoC?H2-Cre cells by RNA-Seq. In addition, ChIP-Seq will determine direct targets of MAFB2 and MAFA/MAFB in proliferating and non-proliferating EndoC?H2 cells. Because transcription factors do not regulate gene expression alone, I will identify coregulators of MAFB2 and MAFA/B using a combined immunoprecipitation and mass spectrometry technique. Coregulator interactions will be confirmed in EndoC?H2 cells with multiple approaches, and assayed in healthy and diabetic islets using a single cell based method. I hypothesize that differences in gene regulation and glucose responsiveness due to a unique set of coregulators recruited in the context of immature (MAFB2-containing) versus mature (MAFA/MAFB-containing) ? cells will exist. Taken together, this proposal will define the molecular mechanism by which juvenile and adult ? cells are regulated by MAFB2 and MAFA/B and determine transcription factor:coregulator interactions in physiological and pathophysiological conditions in human islet cells.
(3 sentences) This proposal aims to define the roles of the homo- or heterodimeric acting transcription factors MAFA and MAFB in human islets utilizing the human ? cell line, EndoC?H2. In addition, biochemical and mass spectrometry methods and single cell analysis will determine coregulator interactions of MAFB2 (found in juvenile ? and ? cells) and MAFA/MAFB (found in mature ? cells). These findings will then be used to delineate the presence and impact of transcription factor:coregulator interactions to human ? cell activity during physiological and diabetic states.
| Iacovazzo, Donato; Flanagan, Sarah E; Walker, Emily et al. (2018) MAFA missense mutation causes familial insulinomatosis and diabetes mellitus. Proc Natl Acad Sci U S A 115:1027-1032 |
