Abstract
Background
Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor regulating the expression of intestinal epithelial genes. Up to 12 HNF4α isoforms can be produced by the use of promoters P1 and P2, and alternative splicing. HNF4α has recently been associated with colorectal cancer, and the variable expression of the P1 and P2 classes could explain the contradictory roles of HNF4α in this disease. However, little is known on the specific functions for each HNF4α isoform during colorectal cancer.
Aims
The overall aim of this study was to elucidate how HNF4α, through the expression of its many isoforms in the colon, may contribute to the progression of colorectal cancer. More specifically, the twelve HNF4α isoforms were functionally characterized by evaluating their DNA binding and transactivation capabilities, and identifying distinct protein interaction networks using quantitative proteomics.
Methods
Expression of the HNF4α isoforms was evaluated by RT-PCR in different cell lines and in different human gastrointestinal tissues. Stable cell lines expressing each of the twelve isoforms tagged with either GFP or BioID2 proteins were generated in the HCT116 human colorectal cancer cell line. EMSA, luciferase and qPCR assays were performed to characterize every single isoform. Protein interaction networks for specific isoforms expressed in the colon were characterized by quantitative mass spectrometry.
Results
Important variations in the expression of HNF4α isoforms were first observed by RT-PCR in different colorectal cancer cell lines as well as in several healthy human gastrointestinal tissues. Stable HCT116 cell lines expressing each of the HNF4α isoforms in an inducible manner were generated. Although each isoform was expressed and localized in the nucleus with a similar pattern, EMSA assays revealed differences in DNA binding capabilities for a subset of isoforms. This correlated with differences observed in the transactivation potential of these isoforms, as determined by luciferase assays and qPCR. Finally, important variations in the protein interactomes for these different isoforms were detected by quantitative mass spectrometry. The nature of these differences was related to proteins involved in transcription and chromatin assembly regulation.
Conclusions
Our data identified differential transcriptional properties among the various HNF4α isoforms. Specific transcriptional partners were identified to differently interact with these isoforms. It is expected that the future characterization of these specific interactions will provide insight as to why HNF4α could have both oncogenic and tumor suppressive properties during colorectal cancer.
Funding Agencies
CIHRNSERC