Dissecting the role of HNRNPD isoforms in determination of cell identity

Cells may respond to stresses found in their internal and external environment in different ways. In many cases, cells will simply die, but increasing evidence suggests that rather than dying, cells may sometimes evade stress by changing into other cell types. These cell identity changes are often associated with diseases such as diabetes or cancer. We have previously identified that pancreatic beta cells, which secrete insulin and are responsible for maintaining stability of blood glucose, sometimes change into other cell types under conditions of disrupted metabolism, and make a completely different hormone, somatostatin. In very recent unpublished work, we have been able to identify the gene (HNRNPD) which is responsible for this change. HNRNPD is one of a class of genes responsible for the regulation of other genes and has many mechanisms by which it accomplishes this. When genes are activated, they produce an RNA message. The HNRNPD gene actually produces 4 different types of RNA message when it is activated, which have different characteristics and may underpin its multifunctionality. In this project, we plan to use pancreatic beta cells, and another cell type (the epithelial cells of the retina) that frequently undergoes cell identity changes, to uncover which aspect of HNRNPD biology is responsible for effects on cell identity, and how this happens. Firstly, we aim to find out what each different HNRNPD RNA message (isoform) does to the identity of 2 different types of cells that are known to change identity when exposed to stress. Secondly, we aim to identify which downstream genes each HNRNPD isoform regulates, and how it does so. Thirdly, we aim to determine what is different about the cells that change their identity, compared with those in the same population that do not. Finally, we aim to see if we can protect cells from changing their identity by manipulating the levels of different HNRNPD isoforms. This work will allow us to unravel the genetic changes that cause cells to change their identity under disease conditions or exposure to a challenging environment, such as diabetes or ageing.

Cell identity changes are known to occur in response to stressful stimuli, and are commonly associated with disease. We have previously identified that under conditions of cellular stress, pancreatic beta cells can transdifferentiate to somatostatin-secreting delta cells and implicated the Heterogeneous Nuclear Ribonucleoprotein Particle D (HNRNPD) gene in this change. HNRNPD encodes a multifunctional RNA binding protein with roles in alternative splicing, mRNA stability and regulation of translation. Its differential roles are governed by the presence of multiple HNRNPD isoforms, which are known to demonstrate differential responses to cell stress. At present, the mechanistic basis by which HNRNPD elicits cell identity change is not known, or whether loss of HNRNPD influences other transdifferentiation-prone cell types. In this work, we plan to functionally dissect the differential activities of HNRNPD isoforms in pancreatic beta cells, and pigmented retinal epithelial cells by manipulation of HNRNPD isoform levels. We will assess the effects of different isoforms on cell identity, target gene binding, mRNA translation, mRNA stability and alternative splicing. Thirdly, we will characterise the transcriptome of the differentiated population relative to the non-transdifferentiated population in the same culture. Finally, we will determine whether by manipulating patterns of HNRNPD splicing we can protect the cells from stress-induced cell identity changes in 2D culture and 3D retinal organoids. This work will determine whether changes to HNRNPD activity or expression are a wider feature of cell identity choice, the mechanisms by which it can regulate cell identity, the gene targets by which the phenomenon is controlled, and which aspects of HNRNPD biology are involved. Our work has implications for both normal development and homeostasis, as well as disease biology, and may provide specific points of traction for protection of cells against stress.