The Research On The Interactome Of Hepatocyte Nuclear Factors

Hepatocyte Nuclear Factors (HNFs), i.e. a group of liver-enriched transcription factors, engage in the process of hepatic phenotype and function, in which many kinds of coactivators of HNFs are required, by regulating many types of tissue-specific expressions of hepatic genes. In this study, protein complexes of main HNFs were purified and identified. Research has also focused on the biological functions and mechanism of interaction of HMG1/HNF1α.The method of coimmunoprecipitation (CO-IP) coupled with MS was adopted to purify and identify interacting proteins of HNF1α, HNF3α, HNF3β, HNF4αand HNF6. A new procedure was given for evaluating the data confidence from four perspectives including compart, domain, function and retrieving information in relevant database. Among the 67 interaction data obtained in the study, 37 (56%) have the common compart with their bait protein, 19 (28%) have the direct interacting domain with their bait protein, 29 (43%) are involved in the process of transcription, and 42 (62%) have the indirect interaction with their bait protein. The result indicates that the data obtained in the study have high reliability. Following this, the author verified interactions of HNF1a and four proteins, i.e. HMG1, 14-3-3zeta, CBX3, and RANBP1 via CO-IP, proved that the four proteins are able to affect the transcription activity of HNF1αthrough luciferase reporter assay, which can be used as a reference for later research in terms of HNF1αtranscription mechanism.Further, CO-IP and GST pull-down were used in sequence to validate the interaction between HMG1 and HNF1α, discovering that HMG box domain of HMG1 and homeodomain of HNF1αis the fragment where HMG1 interacts with HNF1α. Luciferase reporter assay proves that HMG1 is able to enhance transcription activity of HNF1α; EMSA shows that HMG1 improves the DNA binding of HNF1α, and reveals that HMG1 can regulate HNF1αon condition that two HMG box domains exist together. RNAi decreases the level of HMG1 in vivo, changing the expression of a few HNF1αtarget genes, which demonstrates the interaction between HMG1 and HNF1αis of great significance for the occurrence and development of liver and pancreas disease.