Determine Functional domains of HNF6 responsible for HNF6 - PDX1 interaction

and PDX1 stabilization

Kebo Zhang

Research Abstract:

Diabetes is a prevalent issue in modern society rising up to 9.3 percent as of 2012. Among the
26 million patients afflicted with diabetes, approximately 7 million are undiagnosed. As a whole,
diabetes is a result of either beta cell destruction (type 1) or increased insulin resistance (type 2). Beta
cells make up approximately 65 to 80 percent of pancreatic Islets found in the pancreas and they
provide a vital role in glucose homeostasis through the storage and release of insulin. The Pancreatic
Duodenal Homeobox 1 (PDX-1) is a transcription factor notably discovered by Doris Stoffers and her
colleagues that has a pivotal role in beta cell maturation and development. Furthermore, the PDX1 C-
terminal Inhibiting factor (PCIF-1) is a nuclear factor that interacts with PDX-1 through binding and
promotes PDX-1 degradation. Taking this into account, our research focused on the impact of
Hepatocyte Nuclear Factors (HNF), a group of transcription factors found in the liver that is often
misleading due to their wide ranging functions from glucose metabolic processes to cell differentiation
and liver, spleen, endoderm development. More specifically, our experiment centered on Hepatocyte
Nuclear Factor 6 (HNF-6) and its functions in pancreatic beta cells. Previously, our lab has discovered a
competitive interaction between HNF-6, PCIF-1 and PDX-1. Notably, HNF-6 and PCIF-1 has been shown
to have opposite effects on PDX-1 stability. While HNF-6 increased PDX-1 expression, PCIF-1 served to
decrease PDX-1 Expression. Our research then focuses on HNF-6 as we wish to determine which specific
domain of HNF-6 is responsible for binding to PDX-1 and increasing PDX-1 stabilization.

The HNF6 protein possesses an N-terminal transcriptional activation domain, and C-terminal Cut
domain and homeodomain. Both N-terminal and C-terminal cut domain have been reported involved in
protein-protein interaction. Our initial hypothesis was that either the Cut domain or the Homeodomain
was responsible for HNF6 Mediated PDX1 protein stabilization. To elucidate which domain is
responsible, we amplified specific DNA fragments through Polymerase Chain Reactions (PCR) that
encoded different mutational HNF-6 proteins (Δcut, ΔHomeodomain, Δcut + Homeodomain and Cut +
Homeodomain). Restriction enzymes Xba1 and BamH1 were used to digest the plasmid pCDNA3-HA and
the amplified DNA fragments which were then pooled together for ligation in order to make various new
constructs. The new constructs were then transformed into E-Coli bacteria and tested by enzyme
digestion and sequencing to see if they contained the correct mutations for HNF6 mutants expression. If
successful, we will then transfect the wild type and mutant HNF6 into Eukaryotic cell where the inserted
DNA fragments will transcribe into mRNA and further translated into different mutated protein. The
Eukaryotic cell will be checked to see whether or not the construct produced expected mutated HNF-6
protein and then which domain mutation will abolish PDX-1 stabilization. Based on preliminary data,
there is a possibility that the N-terminal is responsible for the HNF-6 and PDX-1 interaction.